Albumin-based non-covalent complexes and methods of use thereof

ABSTRACT

A non-covalent complex of an albumin molecule and a hydrophobic ligand, compositions containing the same, and methods of use thereof are provided. The present complex may find use in delivering the hydrophobic ligand to microorganisms that have albumin-binding outer surfaces, such as a cell wall.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 62/294,931, filed Feb. 12, 2016, and U.S. ProvisionalPatent Application No. 62/158,670, filed May 8, 2015, which applicationsare incorporated herein by reference in their entireties.

INTRODUCTION

Albumin is the most abundant protein in plasma, accounting for more thanhalf of human plasma protein. It is important for various physiologicalprocesses such as providing colloid osmotic pressure, solubilizing longchain fatty acids, delivery of water insoluble nutrients to cells, andbalancing plasma pH. Albumin naturally accumulates at tumors and sitesof inflammation, a characteristic which can be augmented by the additionof targeting ligands. Albumin has two hydrophobic binding sites, inwhich it can transport a hydrophobic ligand that would normally beinsoluble in water.

Bacterial/fungal cells produce various proteins that bind to albumin andlikely impart survival ability against vertebrate host defensemechanisms and/or virulence to the bacterial cells. Many gram-positivebacteria express surface proteins with ability to bind serum proteins.The surface proteins typically contain repeated tandem serumprotein-binding domains with one or several specificities, which ofteninclude albumin binding. The bacteria can thereby camouflage themselveswith bound host-proteins to evade the immune system and potentially alsoscavenge protein-bound nutrients

Expression of albumin-binding proteins has been shown to promotebacterial growth and virulence. There are many different types ofalbumin-binding proteins with different size and function. For example,more than 40 albumin-binding domains have been found in one protein,forming a rod-like structure in a giant cell wall-associatedfibronectin-binding molecule. Protein G-related albumin-binding (GA)modules occur on the surface of numerous Gram-positive bacterialpathogens and their presence may promote bacterial growth and virulencein mammalian hosts.

SUMMARY

Provided herein are hydrophobic ligand-albumin complexes, and methods ofmaking and using the same. The present hydrophobic ligand-albumincomplexes provide a delivery vehicle for targeting a hydrophobicmolecule to a microorganism, and may find use in the detection, e.g.,optical detection, of microorganisms in a sample and in the formulationof therapeutic compositions containing hydrophobic active agents, e.g.,hydrophobic antibacterial or antifungal agents, for administration to anindividual in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1 is a graph showing the absorption spectrum of concentratedlycopene solution in hexane, extracted from a tetra-hydro furan (THF)solution; and dilute lycopene in human serum albumin (HSA) extractedfrom THF, according to embodiments of the present disclosure.

FIG. 2 is a graph showing an ultraviolet-visible (UV-Vis) spectrum oflycopene in HSA in an aqueous solution as it is first formulated bytransferring from acetone, compared to the spectrum when the lycopene inHSA is dried and resuspended in water, and when the water suspension issonicated using an 800 W sonicator for 10 minutes, according toembodiments of the present disclosure.

FIG. 3 is a graph showing the UV-Vis spectrum of an aqueous solution ofAmphotericin-B incorporated into bovine serum albumin (BSA), accordingto embodiments of the present disclosure.

FIG. 4 is a graph showing the UV-Vis spectra of an aqueous solution ofCamptothecin (CPT) incorporated into bovine serum albumin (BSA),according to embodiments of the present disclosure.

FIG. 5A is a graph showing UV-Vis absorbance spectra of lycopene/HSA inthe presence or absence of Staphylococcus warneri, according toembodiments of the present disclosure.

FIG. 5B is a graph showing UV-Vis absorbance spectra of lycopene/HSA inthe presence or absence of Staphylococcus aureus, according toembodiments of the present disclosure.

FIG. 6 is a graph showing the difference between the UV-Vis absorptionprofiles of initial and final states (defined as Profile A-Profile C inFIG. 5B) for 3 different concentrations of lycopene in HSA for S. aureusat 1.7×10⁶ CFU/mL, according to embodiments of the present disclosure.

FIG. 7 is a graph summarizing binding of HSA to differentmicroorganisms.

FIG. 8 is a graph showing growth curves of Candida albicans (ATCC90028), with an initial concentration of 5×10⁵ CFU/mL under differentconcentrations of Amphotericin B/bovine serum albumin complex (AmpB/BSA)or liposomal Amphotericin B (LAMB), according to embodiments of thepresent disclosure.

FIG. 9 is a graph showing growth curves of Candida glabrata, with aninitial concentration of 5×10⁵ CFU/mL under different concentrations ofAmpB/BSA or LAMB, according to embodiments of the present disclosure.

FIG. 10 is a graph showing growth curves of S. Epidermidis, with aninitial concentration of 5×10⁵ CFU/mL under different concentrations ofClofazamine/BSA or Clofazamine in an organic solvent formulation,according to embodiments of the present disclosure.

FIG. 11 is a collection of graphs showing UV-Vis absorption spectra oftwo lycopene solutions in hexane.

FIG. 12 is a collection of graphs showing changes in the UV-Visabsorption spectrum of overloaded lycopene/HSA upon addition of HSA.

FIG. 13 is a graph showing circular dichroism spectra of two samplescontaining lycophene/HSA, without and with added 1000 CFU/mL Klebsiellapneumoniae, according to embodiments of the present disclosure.

FIG. 14 is a graph showing the difference between the amplitude of thelycopene Raman peak at 1516 cm⁻¹ at time t=0 and t=15 min for a control(uninfected) sample, and one that contains 100 CFU/6 mL S. aureus,according to embodiments of the present disclosure.

FIG. 15 is a collection of graphs showing lycopene Raman peak height asa function of time for a test sample with 100 CFU/6 mL of S. aureus,according to embodiments of the present disclosure.

FIG. 16 is a graph showing the height of the resonant Raman peak at 1156cm⁻¹ as a function of lycopene content for uninfected samples, andsamples that contain 100 CFU/mL S. aureus, according to embodiments ofthe present disclosure.

FIG. 17 is graph showing lycopene Raman peak height as a function ofpathogen concentration, according to embodiments of the presentdisclosure PHS: pooled human serum.

FIG. 18 is a graph showing the rate of change of the Raman peaks withvarying amounts of S. aureus, according to embodiments of the presentdisclosure.

FIG. 19 is a graph showing the negative of the rate of change of thelycopene Raman peak with varying concentrations of vancomycin resistantEnterococci, according to embodiments of the present disclosure.

FIG. 20 is a graph showing the negative of the rate of change of thelycopene Raman peak from different experimental samples with or withoutaddition of 100 CFUs of S. aureus, according to embodiments of thepresent disclosure.

FIG. 21 is a graph showing the rate of change of the lycopene Raman peakfrom clinical samples having known infection status, according toembodiments of the present disclosure.

FIGS. 22A and 22B are a collection of graphs characterizing the spatialprofile of lycopene Raman peak height in a vial, according toembodiments of the present disclosure.

FIG. 23 is a collection of graphs showing a prediction of the minimalinhibitory concentration (MIC) of S. aureus at 200 CFU/mL, according toembodiments of the present disclosure.

FIG. 24 is a graph showing UV-Vis absorption profile difference betweenlycopene/HSA and lycopene/HSA with S. aureus, for 3 differentconcentrations of lycopene.

FIG. 25 provides NCBI Reference Sequence: NP_000468.1 for human serumalbumin preprotein.

FIG. 26 provides NCBI Reference Sequence: NP_851335.1 for bovine serumalbumin precursor.

FIG. 27 provides NCBI Reference Sequence: NP_033784.2 for mouse serumalbumin preprotein.

FIG. 28 provides NCBI Reference Sequence: NP_599153.2 for rat serumalbumin precursor.

FIG. 29 provides NCBI Reference Sequence: XP_005681801.1 for goat serumalbumin (predicted).

FIG. 30 provides NCBI Reference Sequence: NP_001310707.1 for donkeyserum albumin precursor.

FIG. 31 provides NCBI Reference Sequence: NP_001075972.1 for horse serumalbumin precursor.

FIG. 32 provides NCBI Reference Sequence: XP_010967650.1 for camel serumalbumin (predicted).

FIG. 33 provides NCBI Reference Sequence: XP_010981066.1 for camel serumalbumin (predicted).

DEFINITIONS

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or ±10%, e.g., ±5%, ±1%, and including±0.1% from the specified value, as such variations are appropriate toperform the disclosed methods or achieve the desired results.

A “plurality” contains at least 2 members. In certain cases, a pluralitymay have at least 10, at least 100, at least 1000, at least 10,000, atleast 100,000, at least 10⁶, at least 10⁷, at least 10⁸ or at least 10⁹or more members.

A “microorganism” as used herein, may refer to any organism that ischaracterized by having a cell wall. Such organisms may include, withoutlimitation, prokaryotes (such as Bacteria and Archaea) and fungi.

“Complex” as used herein, may refer to two or more entities thatphysically associate with each other, but not with other entities. Thetwo or more entities may be able to migrate or diffuse through a mediumas a single unit.

“Hydrophobic” as used herein, may describe a molecule or compound thatis poorly soluble in water, at least around physiological pH. In somecases, the molecule or compound may have solubility in water of 1.0mg/mL or less, e.g., at 25° C.

“Functionally associate”, as used herein, may be used to describe afirst entity and a second entity physically interacting, directly orindirectly, with each other such that a property of the first entityand/or the second entity is altered as a result of the interaction. Insome cases, the physical interaction may include the first entitybinding to, or forming a complex with, the second entity, or the firstentity being transferred to the second entity.

As used herein, “Raman scattering,” and other similar terms and/orphrases, may refer to any method whereby light incident on a sample at afixed wavelength is scattered at other wavelengths. The scattering maybe by an incoherent process due to the absorption of the incident photonby the excitation of the structure from an initially lower (the groundstate) to a higher vibrational level, and subsequent relaxation down toa different ground state level.

As used herein, “Raman band” and similar terms and/or phrases may referto the spectral profile (e.g. intensity versus frequency) correspondingto the Raman scattering from a particular chemical bond within amolecule. It is understood that each chemical bond manifests as a Ramanband at distinct frequencies and that in some cases, these Raman bandsmay overlap, making them difficult to distinguish. Further, it isunderstood that the Raman cross section of a chemical bond is a“constant” that defines the intensity of the corresponding Raman peak.Furthermore, it is understood that this cross section can change withwavelength of incident light, and/or with optical resonance of theincident light with an absorption band, and/or with changes in theimmediate environment of that chemical bond. Such a resonance changeoccurs during resonant Raman enhancement.

As used herein, it is understood that the “Raman spectrum” of a sample,and similar terms and/or phrases, refer to the sum of all the Ramanbands, and the relative heights on individual Raman bands in a Ramanspectrum is proportional to the relative abundance of the correspondingchemical bonds multiplied by their Raman cross section.

As used herein, “absorption” and similar terms and or phrases refer toany method wherein incident light is absorbed by a sample of interest.The incident photon may interact with a structure by any number ofmechanisms, including the excitation of outer electrons (e.g.corresponding to the absorption of UV or visible radiation), or theexcitation of the molecule into higher vibrational/rotational energystates.

As used herein, “Resonant Raman scattering,” and similar phrases and/orterms, refers to a process that is understood to be a special type ofRaman scattering process that involves the excitation of a molecule froman initial ground state to a real excited state that corresponds to areal vibrational state. Thus, for the purpose of the present discussion,resonant “Raman enhancement” (or “resonance Raman”), and other similarterms and/or phrases, refer to any method whereby the Raman crosssection of a particular band is enhanced by the strong opticalabsorption.

As used herein, “profile” may refer to a set of measurements of aproperty of a sample obtained across one or more dimensions in timeand/or space. A “temporal profile” may be obtained by measuring theproperty over a plurality of time points. A “spatial profile” may beobtained by measuring a property over a plurality of locations. In somecases, the plurality of locations is a plurality of locationssubstantially in one dimension (i.e., substantially along a line inspace).

An “aggregate” as used herein, may refer to a collection of molecules ina liquid medium, wherein the molecular interactions are stable enough todetectably alter a physical property of the system, compared to a systemin which the molecules do not exhibit the interactions among themselvesin the liquid medium. The physical property altered may include anoptical property (e.g., absorbance, Raman spectrum) of the system.

As used herein, “vial” and other similar terms and/or phrases refer to atest container that contains the test sample along with any othercomponents of the assay. It is understood that the vial can beconstructed out of any suitably transparent material, such as glass andplastics.

The terms “polypeptide,” “peptide,” and “protein”, used interchangeablyherein, refer to a polymeric form of amino acids of any length, whichcan include genetically coded and non-genetically coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified peptide backbones. The term includes fusionproteins, including, but not limited to, fusion proteins with aheterologous amino acid sequence, fusions with heterologous andhomologous leader sequences, with or without N-terminal methionineresidues; immunologically tagged proteins; and the like.

Before the present disclosure is further described, it is to beunderstood that the disclosed subject matter is not limited toparticular embodiments described, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present disclosure will belimited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the disclosed subject matter. Theupper and lower limits of these smaller ranges may independently beincluded in the smaller ranges, and are also encompassed within thedisclosed subject matter, subject to any specifically excluded limit inthe stated range. Where the stated range includes one or both of thelimits, ranges excluding either or both of those included limits arealso included in the disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the disclosed subject matter belongs. Although anymethods and materials similar or equivalent to those described hereincan also be used in the practice or testing of the disclosed subjectmatter, the preferred methods and materials are now described. Allpublications mentioned herein are incorporated herein by reference todisclose and describe the methods and/or materials in connection withwhich the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “ahydrophobic molecule” includes a plurality of such hydrophobic moleculesand reference to “the albumin protein” includes reference to one or morealbumin proteins and equivalents thereof known to those skilled in theart, and so forth. It is further noted that the claims may be drafted toexclude any element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

It is appreciated that certain features of the disclosed subject matter,which are, for clarity, described in the context of separateembodiments, may also be provided in combination in a single embodiment.Conversely, various features of the disclosed subject matter, which are,for brevity, described in the context of a single embodiment, may alsobe provided separately or in any suitable sub-combination. Allcombinations of the embodiments pertaining to the disclosure arespecifically embraced by the disclosed subject matter and are disclosedherein just as if each and every combination was individually andexplicitly disclosed. In addition, all sub-combinations of the variousembodiments and elements thereof are also specifically embraced by thepresent disclosure and are disclosed herein just as if each and everysuch sub-combination was individually and explicitly disclosed herein.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the disclosed subjectmatter is not entitled to antedate such publication by virtue of priordisclosure. Further, the dates of publication provided may be differentfrom the actual publication dates which may need to be independentlyconfirmed.

DETAILED DESCRIPTION

As summarized above, hydrophobic ligand-albumin complexes, and methodsof making and using the same are provided. The present hydrophobicligand-albumin complexes may retain many properties of albumin inuncomplexed form, such as the ability to stay unaggregated under normaloperation and the ability to aggregate under specific conditions. Thehydrophobic ligand-albumin complex may remain soluble in aqueoussolution as prepared, and may provide an efficient way to deliver ahydrophobic molecule that is not normally soluble in aqueous solution toa target, e.g., an albumin-binding target, in aqueous solution. Manyinfectious or opportunistic microorganisms express albumin-bindingmoieties on the cell surface, e.g., on the surface of the cell wall.Thus, the hydrophobic ligand-albumin complex can deliver the hydrophobicmolecule in complex with albumin to microorganisms. A hydrophobicligand-albumin of the present disclosure may find use in detectingmicroorganisms in a sample, e.g., a clinical sample, or to enhance theefficacy of antimicrobial compounds. For example, in the case of ahydrophobic antimicrobial compounds, a complex of an antimicrobialcompound with the albumin may provide: (a) effective solubilization ofthe antimicrobial agent, such that it can be transported effectively;(b) preferential transport of the antimicrobial to the pathogen (asopposed to enhanced transport in a random direction); and (c) depositionof the antimicrobial agent on the surface of the pathogenicmicroorganism.

Further aspects of the present disclosure are now described.

Hydrophobic L Ligand-Albumin Complexes

Provided herein is a non-covalent complex of a hydrophobicmolecule/ligand and an albumin protein, where the interaction betweenthe hydrophobic ligand and the albumin in the complex does not include acovalent bond. A complex of the present disclosure does not include anaggregate of two or more albumin protein molecules, such asnanoparticles of albumin. However, multiple complexes of the presentdisclosure, each containing an albumin protein, may form an aggregate insolution under certain circumstances, as described herein.

The albumin protein may be any suitable albumin. Suitable albuminproteins include, but are not limited to, human serum albumin (HSA; GeneID: 213); bovine serum albumin (BSA; Gene ID: 280717); mouse albumin(Gene ID: 11657); rat albumin (Gene ID: 24186); goat albumin (Gene ID:100860821); donkey albumin (Gene ID: 106835108); horse albumin (Gene ID:100034206); camel albumin (Gene ID: 105080389 or 105091295), etc. Thealbumin protein may also include any albumin variants suitable for usein a hydrophobic ligand-albumin complex.

In some embodiments, a suitable human serum albumin protein comprises anamino acid sequence having at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to amino acids 25-609 of theamino acid sequence depicted in FIG. 25 (SEQ ID NO:1).

In some embodiments, a suitable bovine serum albumin protein comprisesan amino acid sequence having at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to amino acids 25-607 of theamino acid sequence depicted in FIG. 26 (SEQ ID NO:2).

In some embodiments, a suitable mouse serum albumin protein comprises anamino acid sequence having at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to amino acids 25-608 of theamino acid sequence depicted in FIG. 27 (SEQ ID NO:3).

In some embodiments, a suitable rat serum albumin protein comprises anamino acid sequence having at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to amino acids 25-608 of theamino acid sequence depicted in FIG. 28 (SEQ ID NO:4).

In some embodiments, a suitable goat serum albumin protein comprises anamino acid sequence having at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to amino acids 25-607 of theamino acid sequence depicted in FIG. 29 (SEQ ID NO:5).

In some embodiments, a suitable donkey serum albumin protein comprisesan amino acid sequence having at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to amino acids 25-607 of theamino acid sequence depicted in FIG. 30 (SEQ ID NO:6).

In some embodiments, a suitable horse serum albumin protein comprises anamino acid sequence having at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to amino acids 25-607 of theamino acid sequence depicted in FIG. 31 (SEQ ID NO:7).

In some embodiments, a suitable camel serum albumin protein comprises anamino acid sequence having at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to amino acids 25-607 of theamino acid sequence depicted in FIG. 32 (SEQ ID NO:8).

In some embodiments, a suitable camel albumin protein comprises an aminoacid sequence having at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, at least about 98%, at least about 99%,or 100%, amino acid sequence identity to amino acids 25-607 of the aminoacid sequence depicted in FIG. 33 (SEQ ID NO:9).

The hydrophobic ligand of the present complex may be any suitablehydrophobic molecule that can bind to an albumin molecule whilesubstantially maintaining desired properties of albumin molecule, e.g.,solubility, binding ability to other albumin molecules and/or albuminreceptors, etc. In some cases, a suitable hydrophobic molecule can bindto an albumin molecule without significantly altering the interactionwith other albumin molecules that would in turn significantly alteraggregation of albumin in solution.

The hydrophobic molecule may have any suitable molecular weight to binda hydrophobic binding site of albumin. In some cases, the hydrophobicmolecule has a molecular weight of 100 kD or less, e.g., 50 kD or less,20 kD or less, 10 kD or less, 5.0 kD or less, including 1.0 kD or less,and has a molecular weight of 0.05 kD or more, e.g., 0.1 kD or more, 0.2kD or more, 0.3 kD or more, including 0.5 kD or more. In someembodiments, the hydrophobic molecule has a molecular weight in therange of 0.05 kD to 100 kD, e.g., 0.05 kD to 50 kD, 0.1 kD to 20 kD,including 0.1 kD to 10 kD. Binding of a hydrophobic molecule to albumincan be measured using any suitable method, such as those describedherein, and by competition assays with known albumin binding agents(see, e.g., US 20150309040, which is incorporated herein by reference),or any other suitable method.

In some cases, the hydrophobic molecule includes a chromophore, e.g., achromophore whose optical property is concentration-dependent due to anoptical interaction between adjacent molecules in close proximity. Thechromophore concentration-dependent optical property may be any suitableoptical property for detecting a change in the aggregation status of thechromophore-containing molecule. In some cases, the absorbance of thechromophore is altered (e.g., red-shifted or blue shifted) in aconcentration-dependent manner due to optical interactions betweenmolecules that contain the chromophore. In some cases, the Ramanscattering of the chromophore is altered (e.g., more or less efficient)in a concentration-dependent manner due to optical interactions betweenmolecules that contain the chromophore.

Suitable chromophore-containing molecules include, but are not limitedto, carotenoids. carotenoids of interest include, but are not limitedto, carotene (e.g., α-carotene, β-carotene, γ-carotene, δ-carotene,δ-carotene, lycopene, etc.) and xanthophylls (e.g., lutein, zeaxanthin,neoxanthin, violaxanthin, flavoxanthin, α- and β-cryptoxanthin, etc.).

In some cases, suitable carotenoids are those compounds that have astrong resonance Raman peak. For example, excitation of many carotenoidswith monochromatic light induces prominent resonance Raman peaks atwavenumbers around 1520 cm⁻¹ and around 1160 cm⁻¹ (see, e.g., Merlin.Pure and Applied Chemistry 57.5 (1985): 785-792). Specifically forlycopene, the Raman spectrum of lycopene excited at a wavelength of 532nm includes two strong peaks at 1516 and 1156 cm⁻¹ (see, e.g., Hoskins,Journal of Chemical Education 61, no. 5 (1984): 460; and López-Ramírezet al., Journal of Raman Spectroscopy 41.10 (2010): 1170-1177). Thepeaks at 1516 and 1156 cm⁻¹ correspond to the v(C═C) and v(C—C)vibrations typical of conjugated polyenes, and are referred to as v₁ andv₂ modes.

In some cases, the hydrophobic molecule includes a fluorophore, e.g., afluorophore whose optical property is concentration-dependent due to anoptical interaction between adjacent molecules in close proximity. Thefluorophore concentration-dependent optical property may be any suitableoptical property for detecting a change in the aggregation status of thefluorophore. In some cases, the hydrophobic molecule is one of a Försterresonance energy transfer (FRET) pair of fluorescent molecules. In sucha case, the hydrophobic molecule may be a donor or an acceptor of theFRET pair, such as DiIC₁₈(3) (DiI) and DiOC18(3) (DiO).

In some embodiments, the hydrophobic molecule is an antimicrobial agent.The antimicrobial agent may be any suitable hydrophobic compound withantimicrobial activity. In some cases, the antimicrobial agent is anantibacterial (antibiotic) or an antifungal agent. Suitableantibacterial agents include, without limitation, clofazimine,chlorhexidine, tetracycline, tobramycin, and gentamicin. Suitableantifungal agents include, without limitation, amphotericin B,pimaricin, filipin, nystatin, itraconazole, ketoconazole, fluconazole,saperconazole, miconazole, ravunconazole, posaconazole, voriconazole,ciclopirox olamine, butoconazole and tolnaftate.

In some cases, the antimicrobial agent is a pharmacologically activeagent. Any suitable hydrophobic pharmacologically active agent may becomplexed with albumin. In some cases, the pharmacologically activeagent is an anti-cancer drug, an anti-viral drug or a cardiovasculardrug. Suitable anti-cancer drugs include, without limitation,camptothecin, silatecan 7-t-butyldimethylsilyl-10-hydroxycamptothecin(DB-67), 7-ethyl-10-hydroxy-20(S)-camptothecin (SN-38), topotecan,irinotecan, 9-nitro-camptothecin, lurtotecan, exatecan, gimatecan,karenitecin, paclitaxel, 5-fluorouracil, prednisone,medroxyprogesterone, megestrol, diethylstilbestrol, melphalan andchlorambucil. Suitable anti-viral drugs include, without limitation,disoxaril, adefovir, maraviroc, dipivoxil, delavirdine, efavirenz,nevirapine, darunavir, amprenavir and tipranavir. Suitablecardiovascular drugs include, without limitation, gemfibrozil,tetrahydrolipstatin, cholestyramine, colestipol, lovastatin, probucol,and squalene.

The hydrophobic ligand-albumin complex of the present disclosure may besoluble in aqueous solution, as prepared by a method described herein.Thus, in some cases, incorporation of the hydrophobic molecule inalbumin does not perturb the molecular interactions between albuminsufficiently to cause aggregation of albumin at standard conditions(e.g., at standard temperature and pressure (STP), or at physiologicalconditions).

While albumin can incorporate hydrophobic ligands, the incorporation ofhydrophobic ligands may alter the solubility of the albumin in a mannerthat may enable density fluctuations. As examples, lycopene andβ-carotene are considered as hydrophobic ligands. The phase separationof albumins with and without lycopene and beta carotene can beunderstood with 4 terms: (a) HSA/Lyc-HSA interaction parameter (χ₁₂),(b) HSA/Lyc-water and HSA-water interaction parameters (χ_(1w), χ_(2w)),(c) molecular weights of the two protein molecules, and (d) conformationstates of the two proteins.

From the Flory-Huggins solution theory, the critical interactionparameter, χ_(C), for a binary mixture of polymers is 0 (two componentswill be miscible only if their interaction parameter is below χ_(C)).However, in a ternary system with a solvent, if the solvent (water inthe case of albumin in serum with and without a ligand) is equally goodfor both proteins |χ_(1w)−χ_(2w)|=0; then the two proteins can betotally miscible solution in spite of a small positive value of χ₁₂.Likewise, two proteins in water may be incompatible if they have adifferent interaction with water |χ_(1w)−χ_(2w)|; with a difference ofas little as 0.03 sufficing for incompatibility, and the threshold forphase separation being lowered as |χ_(1w)−χ_(2w)| increases.

The albumin-water interaction parameters χ_(1w) and χ_(2w) can beestimated from the Hildebrand solubility parameters δ.

$\chi_{pw} = {\frac{V_{o}}{RT}\left( {\delta_{p} - \delta_{w}} \right)^{2}}$

Where Vo is the molar volume of the solvent (water). In turn, δ can becalculated from the group contribution method of Van Krevelen.

When comparing albumin with and without lycopene (as an example of ahydrophobic ligand ˜ the values will be very similar for β-carotene),the Hildebrand parameters δ for water, albumin and β-carotene (are 43,23.31 and 14.29 J^(1/2) cm^(−3/2), respectively) are used; and usingthose values, δ₂=23.31 for HSA and δ₁=23.24 for HSA/lyc J^(1/2)cm^(−3/2). With these numbers, χ_(1w)=2.81 and χ_(2w)=2.79, and|χ_(1w)−χ_(2w)|=0.02.

It is noted that this difference is just below the previously notedthreshold for incompatibility between two proteins in water. Thus, whileoutright phase separation is unlikely in the absence of an additionalstimulus, fluctuations in lycopene density is possible/likely. Further,it is noted that albumin has two binding sites; and if it carries twolycopene molecules (one at each binding site), then|χ_(1w)−χ_(2w)|=0.04; which is above the threshold of 0.03. Thus thedouble filled albumin may phase separate into aggregates.

These density fluctuations may result in scattering if the length scaleof these fluctuations is comparable to the length scale of light, and ifthe light is tuned such that it is absorbed only by the lycopene. Thus,depending on the magnitude and length scale of these fluctuations, afinite and variable number of lycopene dense pockets may be observed bythe collecting lens and it may appear that the observed lycopene levelsare quantized, and that these levels are reduced below the expectedvalues.

A single albumin molecule may be complexed with any suitable number ofhydrophobic ligands based, e.g., on the hydrophobicity of the ligands.In some cases, the albumin is complexed with a single hydrophobic ligand(single filled). In some cases, the albumin is complexed with twohydrophobic ligands (double filled). The extent of the number ofhydrophobic ligands on a single albumin protein may be controlled asdescribed further below.

Compositions

Also provided herein are compositions that include a hydrophobicligand-albumin complex, as described above. A composition of interestincludes an aqueous solution that includes an amount of the presenthydrophobic ligand-albumin complex. The hydrophobic ligand-albumincomplex may be in solution (i.e., does not form aggregates), e.g., whenthe composition is initially prepared, or reconstituted from a powderfrom, as described herein. The present composition may find use inassays performed in vitro, e.g., to detect the presence of bacteria in aclinical sample, or in delivering a hydrophobic ligand to a site invivo, e.g., to administer a pharmaceutically active agent to anindividual. As such, the present composition may include any othersuitable components for its intended use.

The composition may include any suitable amount of the hydrophobicligand-albumin complex. In some cases, the composition includes 1.0 nMor more, e.g., 5.0 nM or more, 10 nM or more, 50 nM or more, 100 nM ormore, 0.5 μM or more, 1.0 μM or more, including 5.0 μM or more, andincludes 10 mM or less, e.g., 5.0 mM or less, 1.0 mM or less, 0.5 mM orless, 0.1 mM or less, 50 μM or less, 10 μM or less, including 5.0 μM orless, of the hydrophobic ligand complexed with albumin, as measuredbased on the amount of the hydrophobic ligand in the composition. Insome embodiments, the composition includes a concentration of thehydrophobic ligand complexed with albumin in the range of 1.0 nM to 10mM, e.g., 5.0 nM to 5.0 mM, 10 nM to 1.0 mM, 50 nM to 0.5 mM, 100 nM to0.1 mM, including 0.5 μM to 50 μM, as measured based on the amount ofthe hydrophobic ligand composition.

The aqueous solution may include any suitable water-based solution. Insome cases, the aqueous solution is water. In some cases, the aqueoussolution is a buffer, which may include any suitable buffering agent(i.e., pH controlling agents), such as, but not limited to, phosphate,bicarbonate, citrate, tris(hydroxymethyl)aminomethane (Tris),N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), bicine,tricine, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),3-(N-morpholino)propanesulfonic acid (MOPS),2-(N-morpholino)ethanesulfonic acid (MES) andpiperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES). The buffer mayinclude any suitable components, such as minerals/salts, antioxidants(such as, e.g., ascorbic acid), chelating agents (such as, e.g.,ethylenediaminetetraacetic acid (EDTA) or glutathione), amino acids(such as, e.g., glycine), proteins, preservatives, tonicity controllingagents, and the like. A suitable buffer includes, but is not limited to,phosphate-buffered saline (PBS).

The pH of the aqueous solution may be any suitable pH. In some cases,the pH of the aqueous solution is greater than 5.4, e.g., 5.5 or higher,6.0 or higher, 6.5 or higher, 7.0 or higher, 7.5 or higher, 8.0 orhigher, including 8.5 or higher, and may be 10.0 or less, e.g., 9.5 orless, 9.0 or less, 8.5 or less, including 8.0 or less. In some cases,the pH of the aqueous solution is in the range of 5.5 to 10.0, e.g., 6.0to 9.5, 6.5 to 9.0, 6.5 to 8.5, including 6.5 to 8.0.

In some cases, the composition includes an antioxidant, e.g., ascorbicacid. The antioxidant may be present in any suitable amount. In somecases, the antioxidant is present in the composition at 0.001 mg/mL ormore, e.g., 0.005 mg/mL or more, 0.01 mg/mL or more, 0.02 mg/mL or more,0.05 mg/mL or more, including 0.1 mg/mL or more, and at 10 mg/mL orless, e.g., 1.0 mg/mL or less, 0.5 mg/mL or less, 0.1 mg/mL or less,including 0.05 mg/mL or less. In some embodiments, the antioxidant ispresent in the composition at a concentration in the range of 0.001 to10 mg/mL, e.g., 0.005 to 1.0 mg/mL, 0.01 to 0.5 mg/mL, 0.01 to 0.1mg/mL, including 0.01 to 0.05 mg/mL.

In some embodiments, the composition includes a plurality of hydrophobicmolecules that have properties, e.g., optical properties, that depend onthe intermolecular distance between the hydrophobic molecules, where thecomposition finds use in detecting microorganisms in a sample, asdescribed further below. In such cases, the composition may furtherinclude a nutrition source that can sustain at least some level ofmetabolism of the microorganism. The nutrition source may be anysuitable nutritional medium. In some cases, the nutritional source istrypticase soy broth (TSB), Luria-Bertani (LB) broth, nutrient broth,brain heart infusion broth (BHI), heart infusion broth, M9 broth,peptone water, SOC broth, terrific broth and vegitone.

In some cases, the composition is a therapeutic composition that issuitable for administering to an individual, e.g., to deliver ahydrophobic pharmacological agent to the individual. The hydrophobicpharmacological agent in such a therapeutic composition may be anysuitable therapeutic compound, such as an antimicrobial, antiviral,anti-cancer, anti-inflammatory or a cardiovascular agent, as describedabove.

The therapeutic composition may contain the hydrophobic ligand-albumincomplex in a pharmacological acceptable carrier or excipient. As usedherein, “carrier” or “excipient” includes any and all solvents,diluents, buffers (such as, e.g., neutral buffered saline or phosphatebuffered saline), solubilisers, colloids, dispersion media, vehicles,fillers, chelating agents (such as, e.g., ethylenediaminetetraaceticacid (EDTA) or glutathione), amino acids (such as, e.g., glycine),proteins, disintegrants, binders, lubricants, wetting agents,emulsifiers, sweeteners, colorants, flavorings, aromatisers, thickeners,agents for achieving a depot effect, coatings, antibacterial andantifungal agents, preservatives, antioxidants, tonicity controllingagents, absorption delaying agents, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active substance, its use in the therapeutic compositions maybe contemplated. By “pharmaceutically acceptable” is meant a materialthat is not biologically or otherwise undesirable, i.e., the materialmay be incorporated into a pharmaceutical composition administered to anindividual without causing any undesirable biological effects orinteracting in a deleterious manner with any of the other components ofthe composition in which it is contained. When the term“pharmaceutically acceptable” is used to refer to a pharmaceuticalcarrier or excipient, it is implied that the carrier or excipient hasmet the required standards of toxicological and manufacturing testing orthat it is included on the Inactive Ingredient Guide prepared by theU.S. Food and Drug administration.

In some cases, a therapeutic composition further includes a secondactive agent. The second active agent may be any suitable pharmaceuticalagent. In some cases, the second active agent is an antimicrobial,antiviral, anti-cancer, anti-inflammatory or a cardiovascular agent thatis more soluble and/or has a better pharmacokinetic (PK) profile thanthe hydrophobic molecule.

A composition of the present disclosure further includes a substantiallydry hydrophobic ligand-albumin complex in sheet or powder form. Thesubstantially dry hydrophobic ligand-albumin complex retains itssolubility of the hydrophobic ligand-albumin complex prior to the dryingwhen reconstituted in a suitable aqueous solution, e.g., a buffer, asdescribed above. The substantially dry hydrophobic ligand-albumincomplex may be obtained from an aqueous composition containing thesoluble hydrophobic ligand-albumin complex, as described above, by anysuitable drying method that preserves the functional properties of thealbumin and hydrophobic ligand when reconstituted. In some cases, thesubstantially dry hydrophobic ligand-albumin complex is produced byfreeze drying an aqueous composition containing the soluble hydrophobicligand-albumin complex. In other cases, the substantially dryhydrophobic ligand-albumin complex is produced by lowering the pH of thesolution to the pH that is close to the isoelectric pH of the hostalbumin (ie, the pH at which the surface charge on the albumin is closeto 0), whereby the ligand-albumin complex forms aggregates that crashout of solution, and the solution is then either decanted or filteredoff.

Methods

Method of Making a Hydrophobic Ligand-Albumin Complex

Further provided herein is a method of forming a non-covalent complex ofa hydrophobic molecule and an albumin protein in solution. In generalterms, the method includes dissolving the hydrophobic molecule in asuitable organic solvent to form a first solution; combining the firstsolution with a second solution to provide a third solution, wherein thesecond solution is an aqueous solution of albumin; and removing theorganic solvent from the third solution to provide a fourth aqueoussolution which contains the non-covalent complex of the hydrophobicmolecule and a single albumin protein. The organic solvent of the firstsolution includes at least an organic compound that has solubility,miscibility with water, presence and/or distribution of polar groups,and/or an ability to alter albumin conformation that is similar toacetone. The organic compound may be a ketone-containing compound, suchas an aliphatic ketone that includes 3-5 carbon atoms (i.e., a C₃-C₅ketone), such as, but not limited to, acetone, methyl ethyl ketone,2-pentanone, and 3-pentanone. The albumin in the second solution may bedissolved, i.e., not aggregated, in the aqueous solution.

In some cases, the hydrophobic molecule is soluble in the C₃-C₅ketone-containing compound, e.g., it is soluble in acetone. For suchcases, in some embodiments, the hydrophobic molecule is dissolved in afirst organic solvent that is the C₃-C₅ ketone-containing compound, e.g.acetone. In some cases, the hydrophobic molecule is not soluble in theC₃-C₅ ketone-containing compound, e.g., not soluble in acetone, but isat least partially soluble in a second organic solvent. For such cases,in some embodiments, the hydrophobic molecule is dissolved in a firstorganic solvent that contains a mixture of the C₃-C₅ ketone-containingcompound as well as the second organic solvent. The second organicsolvent may include any suitable organic compound that can dissolve thehydrophobic molecule, is more volatile than water, and is miscible withthe ketone-containing compound, e.g., miscible with acetone. The secondorganic solvent may include, without limitation, methanol, ethanol,dichloromethane, acetonitrile, benzene, n-butanol, butyl acetate, carbontetrachloride, chloroform, cyclohexane, 1,2-dichloroethane, dioxane,ethyl acetate, diethyl ether, heptane, hexane, methyl-t-butyl ether,2-butanone, pentane, n-propanol, isopropanol, diisopropyl ether,tetrahydrofuran, toluene, trichloroethylene, and combinations thereof.In some cases, the hydrophobic molecule is initially dissolved in asecond organic solvent containing one or more of the second organiccompounds. Then the second organic solvent is combined with the C₃-C₅ketone-containing compound, e.g., acetone.

The ratio of the first organic solvent to the second organic solvent maybe any suitable ratio to provide for the hydrophobic ligand-albumincomplex using the present method. In some cases, the ratio of the firstorganic solvent to the second organic solvent used to provide the firstsolution is about 0.001:1 or greater, e.g., about 0.01:1 or greater,about 0.1:1 or greater, about 0.2:1 or greater, including about 1:1 orgreater, and is about 1,000:1 or less, about 100:1 or less, about 10:1or less, about 5:1 or less, including about 1:1 or less, by volume. Insome embodiments, the ratio of the first organic solvent to the secondorganic solvent is in the range from about 0.001:1 to about 1,000:1,e.g., from about 0.01:1 to about 100:1, from about 0.1:1 to about 10:1,including from about 0.2:1 to about 5:1, by volume. In some cases, theratio of the first organic solvent to the second organic solvent isabout 2:1.

The amount of hydrophobic molecule present in the first solution mayvary, depending on the nature of the hydrophobic molecule and thedesired outcome. In some cases, the hydrophobic molecule is present inthe first solution at a concentration of 0.001 mg/mL or more, e.g.,0.005 mg/mL or more, 0.01 mg/mL or more, 0.05 mg/mL or more, 0.1 mg/mLor more, 0.5 mg/mL or more, including 1.0 mg/mL or more, and at aconcentration of 50 mg/mL or less, e.g., 25 mg/mL or less, 10 mg/mL orless, 5.0 mg/mL or less, including 3 mg/mL or less. In some embodiments,the hydrophobic molecule is present in the first solution at aconcentration in the range of 0.001 to 50 mg/mL, e.g., 0.01 to 25 mg/mL,0.05 to 10 mg/mL, including 0.1 to 5.0 mg/mL.

The second solution may include albumin in any suitable amount. Thesecond solution may contain albumin at a concentration of 0.1 mg/mL ormore, e.g., 0.5 mg/mL or more, 1.0 mg/mL or more, 5.0 mg/mL or more, 10mg/mL or more, including 20 mg/mL or more, and at a concentration of 100mg/mL or less, 50 mg/mL or less, 30 mg/mL or less, 15 mg/mL or less,including 10 mg/mL or less. In some embodiments, the second solution maycontain albumin at a concentration in the range of 0.1 to 100 mg/mL,e.g., 0.5 to 50 mg/mL, 1.0 to 30 mg/mL, including 5.0 to 30 mg/mL.

The second solution may include one or more additional components, suchas a buffering agent, (i.e., pH controlling agents), such as, but notlimited to, phosphate, bicarbonate, citrate,tris(hydroxymethyl)aminomethane (Tris),N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), bicine,tricine, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),3-(N-morpholino)propanesulfonic acid (MOPS),2-(N-morpholino)ethanesulfonic acid (MES) andpiperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES). Other suitableadditional components include, without limitation, minerals/salts,antioxidants (such as, e.g., ascorbic acid), chelating agents (such as,e.g., ethylenediaminetetraacetic acid (EDTA) or glutathione), aminoacids (such as, e.g., glycine), proteins, preservatives, tonicitycontrolling agents, and the like. In some cases, the second solutionincludes phosphate-buffered saline (PBS). In some cases, the secondsolution includes a compound that modulates albumin conformation such ashydrophobic free radical scavengers (e.g.2,6-di-tert-butyl-4-methylphenol, Vitamin E).

The pH of the second aqueous solution may be any suitable pH. In somecases, the pH of the aqueous solution is greater than 5.4, e.g., 5.5 orhigher, 6.0 or higher, 6.5 or higher, 7.0 or higher, 7.5 or higher, 8.0or higher, including 8.5 or higher, and may be 10.0 or less, e.g., 9.5or less, 9.0 or less, 8.5 or less, including 8.0 or less. In some cases,the pH of the aqueous solution is in the range of 5.5 to 10.0, e.g., 6.0to 9.5, 6.5 to 9.0, 6.5 to 8.5, including 6.5 to 8.0.

In some cases, the second aqueous solution includes an antioxidant,e.g., ascorbic acid. The antioxidant may be present in any suitableamount. In some cases, the antioxidant is present in the second aqueoussolution at 0.001 mg/mL or more, e.g., 0.005 mg/mL or more, 0.01 mg/mLor more, 0.02 mg/mL or more, 0.05 mg/mL or more, including 0.1 mg/mL ormore, and at 10 mg/mL or less, e.g., 1.0 mg/mL or less, 0.5 mg/mL orless, 0.1 mg/mL or less, including 0.05 mg/mL or less. In someembodiments, the antioxidant is present in the second aqueous solutionat a concentration in the range of 0.001 to 10 mg/mL, e.g., 0.005 to 1.0mg/mL, 0.01 to 0.5 mg/mL, 0.01 to 0.1 mg/mL, including 0.01 to 0.05mg/mL.

The organic solvent (e.g., the C₃-C₅ ketone-containing solvent, such asacetone, or a combination of the C₃-C₅ ketone-containing solvent and asecond solvent) may be removed from the third solution using anysuitable method that retains the functional properties of the albuminand hydrophobic molecule in the complex to provide a fourth solution,wherein the fourth solution is an aqueous solution including anon-covalent complex of the hydrophobic molecule and a single albuminprotein molecule. In some cases, the removing is performed byevaporating the volatile components (e.g., the first organic solventand/or the second organic solvent) from the mixture of the first andsecond solutions.

In some cases, the removing, e.g., evaporating, is performed at lowtemperature and pressure, such as at standard temperature and pressure(STP: 1 atm and room temperature), or any other suitable condition.Evaporation of the organic solvent cools the remaining liquid, which cangradually lower the temperature. In some cases, this may be compensatedfor by sonicating the liquid—the sonication power raises the temperatureof the liquid, and compensates for the lowering of the temperature dueto evaporation. By controlling the sonication power, the temperature ofthe solution can be controlled. In some cases, the evaporating isperformed at a temperature (of the mixture) of 0° C. or more, e.g., 5°C. or more, 10° C. or more, 15° C. or more, 20° C. or more, including30° C. or more, and at a temperature of 40° C. or less, e.g., 38° C. orless, 36° C. or less, 30° C. or less, including 25° C. or less. In somecases, the removing, e.g., evaporating, is performed at a temperature(of the mixture) in the range of 0 to 40° C., e.g., 5 to 38° C., 10 to38° C., 15 to 38° C., including 20 to 38° C. In some cases, theremoving, e.g., evaporating, is performed at an ambient pressure overthe mixture of 1 atm or less, e.g., 0.5 atm or less, 0.2 atm or less,including 0.1 atm or less. In some cases, the removing, e.g.,evaporating, is performed under vacuum pressure. In some cases, theremoving, e.g., evaporating, is performed using a rotary evaporator(rotavap).

In some embodiments, a solution containing albumin (e.g., the secondsolution, the mixture of the first and second solution, and/or the thirdsolution) may be at a suitable temperature for maintaining a desiredalbumin conformation and to provide a desired affinity between thehydrophobic ligand and albumin in the complex. In some cases, a solutioncontaining albumin is maintained at a temperature of about 0° C. ormore, e.g., about 5° C. or more, about 10° C. or more, about 15° C. ormore, about 20° C. or more, including about 30° C. or more, and at atemperature of about 40° C. or less, e.g., about 38° C. or less, about36° C. or less, about 30° C. or less, including about 25° C. or less. Insome cases, a solution containing albumin is at a temperature (of themixture) in the range of about 0 to about 40° C., e.g., about 5 to about38° C., about 10 to about 38° C., about 15 to about 38° C., includingabout 20 to about 38° C.

In some embodiments, the method includes (1) dissolving a hydrophobicmolecule into an organic solvent; (2) mixing this organic solventsolution with acetone in an appropriate ratio (if the ligand isinsoluble in acetone) or drying the organic solvent and reducing theligand to powder form and then redissolving the powder into acetone (ifthe ligand is soluble in acetone); (3) mixing the acetone solution (oracetone-organic solvent mixture solution) with an aqueous solution ofhuman serum albumin; and (4) removing the acetone and other organicsolvents with low boiling points by evaporation.

In the present method, the extent of the number of hydrophobic ligandson a single albumin protein may be controlled by controlling thefollowing factors: (a) molar ratios of the hydrophobic molecule, e.g.,lycopene, to albumin in the third solution; (b) the dilution of albuminin the aqueous solution, where the more concentrated albumin solutionsencourage double filling; (c) the temperature of the transfer process,where lower temperature encourages double filling; and (d) the rate oftransfer of the hydrophobic molecule, e.g., lycopene, from the firstsolution to albumin, where a faster rate encourages double filling.

In some embodiments, the molar ratio of the hydrophobic molecule toalbumin in the third solution is 0.001:1 or more, e.g., 0.005:1 or more,0.01:1 or more, 0.02:1 or more, 0.04:1 or more, including 0.06:1 ormore, and, in some embodiments, is 10:1 or less, e.g., 5:1 or less, 2:1or less, 1:1 or less, 0.5:1 or less, including 0.2:1 or less. In someembodiments, the molar ratio of the hydrophobic molecule to albumin inthe third solution is in the range of 0.001:1 to 10:1, e.g., 0.005:1 to5:1, 0.01:1 to 2:1, 0.02:1 to 1:1, including 0.04:1 to 0.5:1.

For example, in some embodiments, where the hydrophobic molecule islycopene, if the molar ratio is above 0.5:1, then double filled albuminis observed (this manifests as UV-Vis absorption peaks at 565 nm, anoverall red coloration, and a strong background absorption at 600 nm);and if the molar ratio is kept below 0.4:1 then only single filledalbumin is observed (this manifests as the absence of any UV-Vis peaksat 565 nm, an overall orange coloration, and nearly no absorption at 600nm).

The rate of transfer of the hydrophobic molecule, e.g., lycopene, fromthe first solution to albumin may vary, and may depend on: theconcentrations of the hydrophobic molecule in the first solution; therate of addition of the first solution to the second solution; thevigorousness of the mixing (where less vigorous mixing encourages afaster rate of transfer); and the rate of removal of the first solutionfrom the mixture.

Methods of Using a Hydrophobic Ligand-Albumin Complex

Methods of Delivering a Hydrophobic Molecule to a Microorganism

A hydrophobic ligand-albumin complex of the present disclosure finds useas a delivery vehicle to transfer the hydrophobic molecule in thecomplex to a target cell, e.g., a microorganism that has a cell wall,where the hydrophobic molecule becomes associated with themicroorganism. In some cases, the hydrophobic ligand-albumin complextransfers and aggregates the hydrophobic molecule to a cell wall of amicroorganism. Once localized, or transferred, to a microorganism, thehydrophobic molecule may exert its function, depending on the nature ofthe hydrophobic molecule, in or on the microorganism.

An aspect of the present disclosure includes a method of delivering ahydrophobic molecule to the cell wall of a microorganism, the methodincluding contacting the microorganism with an aqueous solutioncontaining a non-covalent complex of a hydrophobic molecule and a singlealbumin protein. It should be noted that the aqueous solution mayinclude many such complexes, wherein each complex is a complex of ahydrophobic molecule or molecules and a single albumin protein molecule.The aqueous solution may be obtained by a method as described herein. Ingeneral terms, the method may include mixing an aqueous solutioncontaining the hydrophobic ligand-albumin complex with a compositionthat contains the microorganism.

The contacting may be performed for a time sufficient to deliver thehydrophobic molecule in the hydrophobic ligand-albumin complex to themicroorganism. In some cases, the aqueous solution containing thehydrophobic ligand-albumin complex is contacted with the microorganismfor 1 min or more, e.g., 5 min or more, 10 min or more, 15 min or more,20 min or more, 30 min or more, including 1 hr or more, and, in somecases, is contacted with the microorganism for 48 hrs or less, e.g., 24hrs or less, 12 hrs or less, 6 hrs or less, 3 hrs or less, 1 hr or less,including 45 minutes or less. In some embodiments, the the aqueoussolution containing the hydrophobic ligand-albumin complex is contactedwith the microorganism for a length time in the range of 1 min to 48hrs, e.g., 5 min to 24 hrs, 10 min to 24 hrs, 10 min to 6 hrs, 10 min to3 hrs, including 10 min to 1 hr.

After the contacting, the method in some cases may include fractionatingthe aqueous solution, e.g, by centrifuging, to separate anymicroorganisms from the bulk solution.

Any suitable amount of the hydrophobic ligand-albumin complex may beused to deliver the hydrophobic molecule to the microorganism. In somecases, the concentration of the hydrophobic molecule complexed toalbumin in the aqueous solution is 1.0 nM or more, e.g., 5.0 nM or more,10 nM or more, 20 nM or more, 50 nM or more, 100 nM or more, 200 nM ormore, 500 nM or more, including 1,000 nM or more, and in someembodiments, is 1.0 mM or less, e.g., 500 μM or less, 200 μM or less,100 μM or less, 50 μM or less, 20 μM or less, 10 μM or less, 5.0 μM orless, 2.0 μM or less, including 1.0 μM or less. In some embodiments, theconcentration of the hydrophobic molecule complexed to albumin in theaqueous solution is in the range of 1.0 nM to 1.0 mM, e.g., 5.0 nM to500 μM, 10 nM to 200 μM, 20 nM to 100 μM, 50 nM to 50 μM, 100 nM to 10μM, including 200 nM to 2.0 μM.

The aqueous solution containing the hydrophobic ligand-albumin complexmay include additional components, such as buffers (e.g., PBS),antioxidants (e.g., ascorbic acid), nutrition sources (e.g., TSB), asdescribed above. Thus, the aqueous solution may include a compositioncontaining the hydrophobic ligand-albumin complex, as described above.

In some cases, the contacting occurs in vitro, e.g., in a vessel, tube,vial, well, multiwell plate, dish, flask, etc. In some cases, thecontacting occurs in vivo, e.g., in an individual who harbors themicroorganism, and to whom a composition that includes the hydrophobicligand-albumin complex has been administered, e.g., using a therapeuticcomposition containing the hydrophobic ligand-albumin complex, asdescribed above. In such cases, the aqueous solution contacting themicroorganism may be a bodily fluid, e.g., blood, plasma, interstitialfluid, lymph, etc., to which the complex is deposited.

The microorganism may be any suitable microorganism that has a cellwall, and that binds albumin. The microorganism may be bacteria orfungi. In some cases, the microorganism is pathogenic or is anopportunistic pathogen. Microorganisms of interest include, withoutlimitation, Escherichia coli 10418, Esch. coli 12241, Staphylococcusaureus, Staph. epidermidis, Klebsiella pneumoniae, Enterobacter cloacae,Enterococcus faecalis, Streptococcus pneumoniae, Pseudomonas aeruginosa,Proteus mirabilis, Strep. pyogenes, Candida albicans, Salmonellatyphimurium, Providencia rettgerri, Bacteroides fragilis, Acinetobacterbaumannii, Enterococcus faecium, Stenotrophomonas maltophilia, Cand.glabrata, Staph aureus (MRSA), and Citrobacter freundii.

Methods of Enhancing Efficacy of Antimicrobial Agents

Where the hydrophobic molecule is an antimicrobial compound, the presentmethod may provide for an efficient method to deliver the antimicrobialcompound to the target microorganism and thereby to reduce or inhibitgrowth of the microorganism. The efficiency of the antimicrobial may bemeasured by a minimum inhibitory concentration (MIC) measured in vitro.In some embodiments, the method reduces the MIC by 10% or more, e.g.,20% or more, 30% or more, 40% or more, including 50% or more, and insome embodiments, the by 99% or less, e.g., 95% or less, 90% or less,80% or less, 70% or less, 60% or less, including 50% or less, comparedto an appropriate control (e.g., the MIC of the antimicrobial compounduncomplexed to albumin tested in otherwise comparable conditions). Insome cases, the method reduces the MIC of the antimicrobial compound bya percentage in the range of 10 to 99%, e.g., 20 to 95%, 20 to 90%, 30to 80%, 40 to 70%, including 40 to 60%, compared to an appropriatecontrol. In some cases, the MIC of the antimicrobial compound used in acomplex with albumin according to a method of the present disclosure hasthe substantially the same MIC as the antimicrobial compound used underan appropriate control condition (e.g., compared to the MIC of theantimicrobial compound not complexed to albumin).

Methods of Determining the Presence of a Microorganism in a Sample

In some cases, the hydrophobic molecule is a compound with one or moredetectable properties, e.g., a compound with detectable opticalproperties, that change significantly depending on intermolecularinteractions, as described above, and the hydrophobic ligand-albumincomplex may be used as a labeling reagent for labeling and detecting amicroorganism in a sample, e.g., a clinical sample, with the hydrophobicligand. The interaction of the metabolically active microorganisms in asample with the albumin of the complex may result in changes in thesample that can be monitored, e.g., monitored optically. For example, inthe baseline state, the hydrophobic molecule, e.g., lycopene,concentration may be substantially uniform throughout the glass vial, asmay be expected if the albumin complex is truly in solution. As themetabolically active bacteria (that may have a concentration of protonson its surface) interacts with the albumin (which may have a negativesurface charge), the albumin may form aggregates. The formation of theseaggregates may result in optical or other changes in the sample,depending on the hydrophobic molecule complexed with the albumin, thatcan be monitored for the presence and/or amount of bacteria.

Thus, in general terms, a microorganism present in the sample may bedetected by first delivering non-covalent complexes, each complexcontaining a non-covalent complex of a hydrophobic molecule and a singlealbumin protein, to the microorganism in the sample of interest, asdescribed above; and then analyzing, e.g., measuring one or moreproperties, e.g., one or more intermolecular distance-dependentproperties, of the sample to determine the presence of themicroorganism. In some embodiments the intermolecular distance-dependentproperties representative of the aggregate form of the hydrophobicmolecule may indicate the presence of the microorganism in the sample;and intermolecular distance-dependent properties representative of thesoluble or non-aggregate form of the hydrophobic molecule may indicatethe absence of the microorganism in the sample.

The following are examples of aqueous solutions that find use in thepresent method of detecting a microorganism in sample:

-   -   0.6 μM lycopene/HSA, 1.2 μM β-carotene/HSA, 2 mL of 1×TSB, 0.02        mg/mL ascorbic acid, and 3.4 mL of PBS with an overall pH of        7.1;    -   1.5 μM lycopene/HSA, 2 mL of 1×TSB, 0.02 mg/mL ascorbic acid,        and 3.4 mL of PBS with an overall pH of 7.5.

The sample tested for the presence of a microorganism by a method of thepresent disclosure may be any suitable sample, such as a body fluidsample, as long as the components of the sample do not substantiallyinterfere with the labeling and detection of the microorganism. In somecases, the sample is a clinical sample, obtained from a healthyindividual, or a patient suspected of having or diagnosed with adisease, e.g., an infectious disease. A suitable sample includes,without limitation, serum, plasma, blood, saliva, mucous, phlegm,cerebral spinal fluid, pleural fluid, tears, lactal duct fluid, lymph,sputum, cerebrospinal fluid, synovial fluid, urine, amniotic fluid, andsemen, etc, and processed forms thereof. In some cases, the sample isprocessed to remove pigmented components, e.g., to remove red bloodcells from blood, using any suitable method. In some cases, the sampleincludes a known amount of a known microorganism. The sample may includeany suitable amount of microorganisms. In some cases, the sampleincludes 1 colony forming units (CFU)/mL or more, e.g., 5 CFU/mL ormore, 10 CFU/mL or more, 100 CFU/mL or more, 1,000 CFU/mL or more, 10⁴CFU/mL or more, 10⁵ CFU/mL or more, including 10⁶ CFU/mL or more, and insome embodiments, includes 10¹⁰ CFU/mL or fewer, e.g., 10⁹ CFU/mL orfewer, 10⁸ CFU/mL or fewer, 10⁷ CFU/mL or fewer, 10⁶ CFU/mL or fewer,including 10⁵ CFU/mL or fewer of the microorganisms. In someembodiments, the sample includes a concentration of the microorganismsin the range of 1 to 10¹⁰ CFU/mL, e.g., 1 to 10⁹ CFU/mL, 1 to 10⁸CFU/mL, 1 to 10⁷ CFU/mL, 1 to 10⁶ CFU/mL, 5 to 10⁵ CFU/mL, 10 to 10⁴CFU/mL, including 10 to 10³ CFU/mL. In some cases, it is not knownwhether or at what concentration microorganisms are present in thesample.

The intermolecular distance-dependent property of the hydrophobicmolecule(s) may be any suitable property. In some cases, theintermolecular distance-dependent property is an optical property, suchas, but not limited to, optical absorbance, Raman scattering,fluorescence, etc. In some cases, the intermolecular distance-dependentproperty is the peak absorbance at optical absorption band. In othercases, the property is the peak of a Raman band. The opticalintermolecular distance-dependent property may be measured using anysuitable method. In some cases, measuring includes using a Ramanspectrometer that resonantly enhances the Raman scattering peak from ahydrophobic molecule, e.g., lycopene (for instance, by using a 532 nmwavelength). In some cases, measuring includes using a UV-Vis absorptionspectrometer (or any other instrument). In some cases, the measuringincludes using a fluorescent microscope, fluorescence spectrometer, orfluorimeter.

In some cases, the method includes measuring the magnitude of an opticalproperty, e.g., a Raman peak height, absorbance at a wavelength, of thesample. For example, a sample labeled with a carotenoid/albumin complex,e.g., a lycopene/HSA complex, may have a resonant Raman peak height uponillumination by a 532 nm wavelength light that is negatively correlatedwith the concentration of a microorganism in the sample. In some cases,the measuring is performed as soon as about 5 minutes, e.g., as soon asabout 10 minutes, after starting the incubation of the microorganismwith the aqueous solution containing the hydrophobic ligand-albumincomplex.

In some cases, the presence or absence of the microorganism in a sampleis determined by measuring a spatial distribution of or temporal changein an optical property of the hydrophobic molecule, or aggregated formthereof, where the optical property is intermoleculardistance-dependent, to obtain a spatial profile or temporal profile,respectively, of the optical property of the sample labeled with thehydrophobic ligand-albumin complex.

In some cases, the method includes measuring the rate of change in anoptical property of the sample, e.g., rate of change of the height of aRaman peak generated by 532 nm light illumination of a sample labeledwith a carotenoid/albumin complex, at a predetermined (or fixed) spatiallocation along the vial or vessel in which the assay is being performed.The predetermined spatial location may be any suitable location alongthe assay vessel. In some cases, the predetermined spatial location is aposition along a vertical dimension of the vessel. The predeterminedspatial location along the vertical dimension of the vessel may be at alevel above where albumin aggregated due to microorganisms segregate andaccumulate. In some cases, the predetermined location is at a distancein the range of about 5 to about 10 mm, about 10 to about 15 mm, about15 to about 20 mm, about 20 to about 25 mm, about 25 to about 30 mm,about 30 to about 35 mm, about 35 to about 40 mm, about 40 to about 45mm, about 45 to about 50 mm, about 50 to about 55 mm, or about 55 toabout 60 mm from the bottom inner surface of the assay vessel.

In some cases, the method includes measuring the spatial distribution ofan optical property, e.g., the height of a Raman peak, along an axis ofthe assay vessel at a predetermined time after mixing the microorganismswith the aqueous solution containing the hydrophobic ligand-albumincomplex. In some cases, the optical property is measured along thevertical axis. In some cases, the optical property is measured acrosslocations in the range of about 0 to about 60 mm, e.g., about 0 to about50 mm, about 0 to about 40 mm, including about 0 to about 30 mm from thebottom inner surface of the assay vessel.

The spatial distribution of or temporal change in an optical property ofthe hydrophobic molecule, or aggregated form thereof, may be at anysuitable time after beginning the assay. In some cases, the opticalproperty is measured at a time point in the range of 1 min to 6 hrs,e.g., 3 min to 3 hrs, 3 min to 1 hr, 3 min to 30 min, including 1 min to5 min, after start of incubation of the microorganisms in the aqueoussolution containing the hydrophobic ligand-albumin complex.

The present method of determining the presence or absence of amicroorganism in a sample may be a rapid method. In some embodiments,the method determines the presence or absence of microorganisms in asample in at most 12 hrs, e.g., at most 6 hrs, at most, 3 hrs, at most 2hrs, including at most 1 hours, from first obtaining the sample, e.g.,clinical sample.

Methods of Measuring the Minimal Inhibitory Concentration ofAntimicrobial Agents

The present methods may also find use in determining the susceptibilityof a microorganism for an antimicrobial. The method may includecombining microorganisms with a plurality of aqueous solutions that eachcontain different concentrations of an antimicrobial agent, in aconcentration range that is expected to cause a concentration-dependentchange in the rate of growth of the microorganisms, ranging fromdecreased or no growth to normal growth (e.g., as determined by growthin the absence of the antimicrobial agent). Thus the aqueous solutionmay contain a nutrition source that supports growth of themicroorganism, in addition to any suitable additional components, asdescribed above. The highest concentration of the antimicrobial at whichthere is at most no growth of the microorganism may correspond to theMIC of the antimicrobial agent for the microorganism.

The microorganism may be obtained from any suitable source. In somecases, the microorganism is obtained from a clinical sample, e.g., fromblood, saliva, mucus, etc., of an individual infected with themicroorganism. In some cases, the MIC for the microorganism is not knownwith respect to the antimicrobial agent. In some cases, the methodincludes growing the microorganism to provide sufficient numbers fordividing into multiple aliquots and testing multiple concentrations ofthe antimicrobial agent.

The present method of determining the MIC of an antimicrobial agent maybe a rapid method. In some embodiments, the present method determinesthe MIC of an antimicrobial agent in at most 12 hours, e.g., at most 10hours, at most 8 hours, at most 6 hours, at most 5 hours, including atmost 4 hours. In some cases, the method is a high-throughput method ofdetermining the MIC of multiple antimicrobial agents for one or moremicroorganisms. Any suitable number of antimicrobial agents may betested. In some cases, the number of antimicrobial agents tested is 2 ormore, e.g., 3 or more, 5 or more, 10 or more, 20 or more, 50 or more,100 or more, including 1,000 or more, and in some embodiments, is100,000 or less, e.g., 10,000 or less, 1,000 or less, including 100 orless.

Methods of Dispersing Albumin Aggregates

Also provided herein is a method of dispersing an aggregate of albumin.The method may include suspending the albumin aggregate in a solutionhaving a pH above 8.0, e.g., 8.2 or higher, 8.4 or higher, 8.6 orhigher, 8.8 or higher, including 9.0 higher, and sonicating thesuspension to disperse the aggregate. In some cases, the albuminaggregate is an aggregate of hydrophobic ligand-albumin complexes, asdescribed above. The pH of the solution may in some cases be 10.0 orlower, e.g., 9.5 or lower, including 9.0 or lower. In some embodiments,the pH of the solution is in the range of 8.2 to 10.0, e.g., 8.2 to 9.5,including 8.2 to 9.0.

The sonicating may be performed for any suitable amount of time. In somecases, the sonication is performed for 5 min or more, e.g., 10 min ormore, including 15 min or more, and in some cases, is performed for 60min or less, e.g., 45 min or less, including 30 min or less. In someembodiments, the sonicating is performed for a duration in the range of5 to 60 min, e.g., 5 to 45 min, including 10 to 30 min.

In some cases, the albumin aggregate may be formed by storing a solutionof dissolved albumin at a temperature below 37° C., e.g., 35° C. orless, 30° C. or less, 20° C. or less, 10° C. or less, including 5° C. orless, and may be formed by storing at a temperature of 0° C. or more,e.g., 5° C. or more, 10° C. or more, 15° C. or more, including 20° C. ormore. In some embodiments, the albumin aggregate is formed by storing asolution of dissolved albumin at a temperature in the range of 0 to 35°C., e.g., 0 to 30° C., 0 to 20° C., including 0 to 10° C.

Kits

Also provided herein is a kit that includes a composition containing anon-covalent complex of a hydrophobic molecule and an albumin protein,as described herein. In some cases, the composition is an aqueouscomposition, or a substantially dry composition. In some cases, the kitfurther includes a buffer that may or may not include one or moreadditional components (e.g., antioxidant, nutrition source, etc.), asdescribed herein.

In some cases, the present kit includes instructions for using acomposition including a non-covalent complex of a hydrophobic moleculeand an albumin protein of the present disclosure. The instructions aregenerally recorded on a suitable recording medium. For example, theinstructions may be printed on a substrate, such as paper or plastic,etc. As such, the instructions may be present in the kits as a packageinsert, in the labeling of the container of the kit or componentsthereof (i.e., associated with the packaging or subpackaging) etc. Inother embodiments, the instructions are present as an electronic storagedata file present on a suitable computer readable storage medium, e.g.CD-ROM, digital versatile disc (DVD), flash drive, Blue-ray Disc™ etc.In yet other embodiments, the actual instructions are not present in thekit, but methods for obtaining the instructions from a remote source,e.g. via the internet, are provided. An example of this embodiment is akit that includes a web address where the instructions can be viewedand/or from which the instructions can be downloaded. As with theinstructions, the methods for obtaining the instructions are recorded ona suitable substrate.

Components of a subject kit can be in separate containers; or can becombined in a single container.

Exemplary Non-Limiting Aspects of the Disclosure

Aspects, including embodiments, of the present subject matter describedabove may be beneficial alone or in combination, with one or more otheraspects or embodiments. Without limiting the foregoing description,certain non-limiting aspects of the disclosure numbered 1-59 areprovided below. As will be apparent to those of skill in the art uponreading this disclosure, each of the individually numbered aspects maybe used or combined with any of the preceding or following individuallynumbered aspects. This is intended to provide support for all suchcombinations of aspects and is not limited to combinations of aspectsexplicitly provided below.

-   1. A method of forming a solution comprising a non-covalent complex    of a hydrophobic molecule and an albumin protein, the method    comprising:    -   dissolving the hydrophobic molecule in:        -   i) a first organic solvent comprising a C₃-C₅ ketone; or        -   ii) a combination of the first organic solvent and a second            organic solvent in a ratio of from about 0.001:1 to about            1000:1 v/v,        -   to provide a first solution;    -   combining the first solution with a second solution to provide a        third solution, wherein the second solution is an aqueous        solution comprising an albumin protein; and    -   removing the first organic solvent or the combination of the        first organic solvent and the second organic solvent from the        third solution to provide a fourth solution,    -   wherein the fourth solution is an aqueous solution comprising a        non-covalent complex of the hydrophobic molecule and a single        albumin protein.-   2. The method of 1, wherein the C₃-C₅ ketone is acetone.-   3. The method of 1 or 2, wherein the hydrophobic molecule is    dissolved in the combination of a first organic solvent and a second    organic solvent, and wherein the ratio of the first organic solvent    and the second organic solvent is in the range of about 1:1 to about    5:1.-   4. The method of 3, wherein the hydrophobic molecule is dissolved in    the combination, and wherein the combination comprises the first    organic solvent and the second organic solvent in a ratio of about    2:1.-   5. The method of any one of 1-4, wherein the hydrophobic molecule is    dissolved in the combination, and wherein the method comprises:    -   dissolving the hydrophobic molecule in the second organic        solvent, to provide a fifth solution; and    -   combining the fifth solution with the C₃-C₅ ketone to provide        the first solution prior to combining the first solution with        the second solution.-   6. The method of any one of 1-5, wherein the removing is performed    by evaporation.-   7. The method of any one of 1-6, wherein the method comprises    contacting a microorganism comprising a cell wall with an aqueous    solution comprising the non-covalent complex of the hydrophobic    molecule and a single albumin protein, wherein the hydrophobic    molecule functionally associates with the microorganism.-   8. The method of 7, wherein the microorganism is a pathogenic    microorganism.-   9. The method of 7 or 8, wherein the contacting occurs in vitro.-   10. The method of 7 or 8, wherein the contacting occurs in vivo.-   11. The method of any one of 1-10, wherein the hydrophobic molecule    is a carotenoid.-   12. The method of 11, wherein the carotenoid is a carotene.-   13. The method of 12, wherein the carotene is lycopene or    β-carotene.-   14. The method of any one of 1-10, wherein the hydrophobic molecule    is an antimicrobial.-   15. The method of 14, wherein the antimicrobial is an antibacterial.-   16. The method of 14, wherein the antimicrobial is an antifungal.-   17. The method of any one of 13-16, wherein the antimicrobial has    increased efficacy when provided in the non-covalent complex    relative to the antimicrobial in an un-complexed state, or when the    antimicrobial is incorporated into other delivery systems.-   18. The method of any one of 1-10, wherein the hydrophobic molecule    is a pharmacologically active agent.-   19. The method of 18, wherein the pharmacologically active agent is    selected from an anti-cancer drug, an anti-viral drug, and a    cardiovascular drug.-   20. The method of any one of 1-19, wherein the albumin protein is a    human serum albumin protein.-   21. The method of any one of 1-19, wherein, the method does not    comprise the use of a potassium phosphate containing reagent.-   22. A method of delivering a hydrophobic molecule to the cell wall    of a microorganism, the method comprising contacting a microorganism    comprising a cell wall with an aqueous solution comprising a    non-covalent complex of a hydrophobic molecule and a single albumin    protein, wherein the hydrophobic molecule functionally associates    with the microorganism.-   23. The method of 22, wherein the microorganism is a pathogenic    microorganism.-   24. The method of 22 or 23, wherein the contacting occurs in vitro.-   25. The method of 22 or 23, wherein the contacting occurs in vivo.-   26. The method of any one of 22-25, wherein the hydrophobic molecule    is a carotenoid.-   27. The method of 26, wherein the carotenoid is a carotene.-   28. The method of 27, wherein the carotene is lycopene or    β-carotene.-   29. The method of any one of 22-25, wherein the hydrophobic molecule    is an antimicrobial.-   30. The method of 29, wherein the antimicrobial is an antibacterial.-   31. The method of 29, wherein the antimicrobial is an antifungal.-   32. The method of any one of 22-31, wherein the albumin protein is a    human serum albumin protein.-   33. A method for determining the presence or absence of a    microorganism in a sample, the method comprising:    -   i) contacting the sample with an aqueous solution comprising a        plurality of non-covalent complexes, each non-covalent complex        comprising a hydrophobic molecule and a single albumin protein,        wherein the hydrophobic molecules are detectable and        functionally associate with a microorganism when present in the        sample;    -   ii) detecting one or more properties of the hydrophobic        molecules; and    -   iii) determining that the microorganism is present or absent in        the sample based on the detecting.-   34. The method of 33, wherein the hydrophobic molecules have one or    more intermolecular distance-dependent properties, wherein the    detecting comprises measuring in the sample one or more profiles of    the one or more intermolecular distance-dependent properties, and    wherein the determining comprises determining that the microorganism    is present when the one or more profiles indicates the presence of    aggregates of the hydrophobic molecule in the sample, or determining    that the microorganism is absent when the one or more profiles    indicates the absence of aggregates of the hydrophobic molecule in    the sample.-   35. The method of any one of 33-34, wherein the microorganism is a    pathogenic microorganism.-   36. The method of 34 or 35, wherein the one or more intermolecular    distance-dependent properties are one or more optical properties.-   37. The method of 36, wherein the one or more intermolecular    distance-dependent properties comprise an optical absorption band    and/or a Raman band.-   38. The method of 36, wherein the one or more intermolecular    distance-dependent properties comprise Förster resonance energy.-   39. The method of any one of 33-37, wherein the hydrophobic molecule    is a carotenoid.-   40. The method of 39, wherein the carotenoid is a carotene.-   41. The method of 40, wherein the carotene is lycopene or    β-carotene.-   42. The method of any one of 34-41, wherein the one or more profiles    comprises a) a temporal profile at a fixed spatial point, and/or b)    a spatial profile at a fixed time point, of the height of Raman    scattered light for the sample obtained by analyzing the sample with    a spectrometer,    -   and wherein the determining comprises determining the presence        or absence the microorganism in the sample based on the height        of one of a plurality of characteristic Ramen peaks in the        spatial profile, and/or the rate of change of one of the        plurality of characteristic Raman peaks in the temporal profile,        relative to a corresponding set of reference profiles.-   43. The method of any one of 33-42, wherein the albumin protein is a    human serum albumin protein.-   44. A method for determining the presence or absence of a    microorganism in a sample, the method comprising:    -   contacting the sample with an aqueous solution comprising a        non-covalent complex of a hydrophobic molecule and a single        albumin protein, wherein the hydrophobic molecule is a        carotenoid and functionally associates with a microorganism,        when present in the sample;    -   illuminating the sample with a broadband light source;    -   collecting and analyzing with a spectrometer light transmitted        through the sample, wherein a temporal profile at a fixed        spatial point, or a spatial profile at a fixed time point, of        the height of a UV-Vis absorption peak is analyzed, and wherein    -   the height of one of a plurality of characteristic Raman peaks        is used as an indicator when compared with a set of control        values, or    -   the rate of change of one of the plurality of characteristic        Raman peaks is used as an indicator, of the presence of the        detectable hydrophobic molecule bound to the microorganism in        the sample.-   45. The method of 44, wherein the microorganism is a pathogenic    microorganism.-   46. The method of 44 or 45, wherein the carotenoid is a carotene.-   47. The method of 46, wherein the carotene is lycopene or    β-carotene.-   48. The method of any one of 44-47, wherein the albumin protein is a    human serum albumin protein.-   49. A composition comprising:    -   an aqueous solution comprising a non-covalent complex of a        hydrophobic molecule and a single albumin protein.-   50. The composition of 49, wherein the hydrophobic molecule is a    carotenoid.-   51. The composition of 50, wherein the carotenoid is a carotene.-   52. The composition of 51, wherein the carotene is lycopene or    β-carotene.-   53. The composition of 49, wherein the hydrophobic molecule is an    antimicrobial.-   54. The composition of 53, wherein the antimicrobial is an    antibacterial.-   55. The composition of 53, wherein the antimicrobial is an    antifungal.-   56. The composition of 53, wherein the hydrophobic molecule is a    pharmacologically active agent.-   57. The composition of 56, wherein the pharmacologically active    agent is selected from an anti-cancer drug, an anti-viral drug, and    a cardiovascular drug.-   58. The composition of any one of 49-57, wherein the albumin protein    is a human serum albumin protein.-   59. A method to disperse aggregates of albumin, the method    comprising:    -   suspending the aggregate in a solution with a pH above 8.0 to        provide a suspension; and/or    -   sonicating the suspension, wherein the aggregate is dispersed.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the disclosed subject matter, and are not intended to limitthe scope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1: Materials and Methods

The following material and methods were used in the Examples, whereapplicable.

Reagents:

The following reagents were purchased from commercial sources listed asfollows: Acetone & hexane: Macron; Ethyl acetate and ethanol: EMD;Pooled human serum: Innovative Research Inc.; Human Serum Albumin HSA:Gemini Bio Products; Lycopene: Sigma-Aldrich; Tryptic Soy Broth TSBpowder: Becton Dickinson; Synthetic defined dropout media powder SDM:Sunrise Science Products. 1× and 4× SDM solutions are 27.49 and 109.96grams, respective, of SDM powder in 1 L of DI water.

Pathogen Stock Solution:

An overnight culture of a known isolate of a particular microorganism ina rich medium (TSB) was centrifuged into a pellet, passaged once for 3hours, centrifuged into a pellet again and then re-suspended in PBS. Theoptical density of the isolate was characterized, and adjusted by addingmore PBS as necessary until OD=0.25 at 600 nm, which was treated as 10⁸CFU/mL (upon checking with an overnight culture on a plate, the actualconcentration was generally found to be within 2× of the inferredconcentration). Serial dilutions was then performed to prepare stocksolutions in PBS at 10¹-10⁸ CFU/mL.

Instrumentation:

For the UV-Vis absorption spectroscopy, METASH Visible Spectrophotometer(en(dot)metash(dot)com/ProductShow(dot)asp?ID=148) was used with a USBinterface. For the circular dichroism measurements, AVIV CircularDichroism Spectrometer, Model 62 DS was used. For the Raman andfluorescence measurements, a commercial spectrometer with a 532 nm/100mW laser, and a CCD that is cooled to −50° C. (vendor: Enwave Optronics,now TSI Inc) were used.

Example 2: β-Carotene Incorporated into Albumin

Aqueous solutions of β-carotene incorporated into human serum albumin(and which can be used to incorporate into other albumins) were preparedusing the following steps: (1) To a 15 mL-centrifuge tube, 23 mg ofβ-carotene (extracted from carrots using hexane; followed by removal ofhexane using evaporation) and 12 mL of acetone were added. The mixturewas vortexed for 1 min and sonicated for 5 min. The mixture was thencentrifuged for 5 min at 4000 RPM, and the yellow solution on top isdecanted off and used for the subsequent steps. (2) To a 500 mL-roundbottom flask, 1.0 gm of commercial human serum albumin HSA, 145 mL ofcommercial PBS buffer, and 0.5 mL of vitamin C PBS buffer solution (10mg/mL) were added. The mixture was shaken well and sonicated for 5 minand used in the next step. (3) After adding a magnetic stirring bar, theHSA solution was stirred vigorously, and β-carotene/acetone solution wasadded slowly via a pipette. After 12 mL of this solution was added, theUV-Vis absorption spectrum of the resulting dark yellow solution wasmonitored. Typically, the absorbance was observed at 456 nm A₄₅₆=2.09.After adding 20 mL of PBS buffer, the resulting mixture was concentratedusing a rotavapor in order to remove all acetone (no bubbling wasobserved during this process and a significant amount of water wascondensed on to the trap surface). The resulting yellow solution wasfiltered through a membrane (200 nm). The UV-Vis spectrum of thefiltered yellow solution was monitored and recorded; in this case, thetypical A₄₅₆=1.50 and final volume was 170 mL. Because β-carotene wasinsoluble in water, and because all organic solvents had been removed inthe final step, the β-carotene was incorporated into the human serumalbumin. Also, based on the UV-Vis absorbance profiles at >600 nm, itwas concluded that the albumin was present in monomeric form(aggregation of albumin results in enhanced Rayleigh scattering, whichcould be detected in the UV-Vis absorbance above 600 nm). Based on theextinction coefficient at 456 nm as 158,000 M⁻¹ cm⁻¹ (obtained throughhexane extraction and UV absorption in hexane with the known extinctioncoefficient as 144,000 M⁻¹ cm⁻¹ at 446 nm), the concentrations wereestimated as [β-carotene]=1.5/158,000=9.49 uM, [HSA]=1000/66000/0.170=90uM. [HSA]/[β-carotene]=90/9.49=, 9.48.

Example 3: Lycopene Incorporated into Albumin

Solutions of lycopene in human serum albumin were prepared using thefollowing steps. Lycopene acetone solution: To a 15 mL-centrifuge tube,28 mg lycopene and 14 mL acetone were added. The mixture was vortexedfor 1 min, sonicated for 5 min and centrifuged for 5 min at 4000 RPM.The reddish solution on top was decanted off and used for the subsequentsteps. HSA/PBS buffer solution: To a 500 mL-round bottom flask, 0.2 gHSA, 200 mL PBS buffer and 0.5 mL ascorbic acid/PBS solution mixture (10mg/mL ascorbic acid concentration) were added. The mixture was shakenwell and sonicated for 5 min. Lycopene/HSA complex solution: Afteradding a magnetic stirring bar, the HSA solution was vigorously stirred,and 16 ml of the lycopene acetone solution was added slowly using apipette. The UV-Vis absorption spectra of the resulting reddish solutionwas monitored, with a typical absorbance A₄₇₆=1.3031, and acetone wasremoved using a rotavapor. The resulting reddish solution was filteredthrough a 0.2 μm membrane; the typical absorbance was A₄₇₆=0.959, andthe typical final volume was about 200 mL. Based on the calculatedextinction coefficient at 476 nm as 155,500 M⁻¹ cm⁻¹ (obtained throughhexane extraction and UV absorption in hexane with the known extinctioncoefficient as 200,000 M⁻¹ cm⁻¹ at 470 nm),[Lycopene]=0.959/155,5000=6.16 μM, [HSA]=1000/66000/0.203=75 μM.[HSA]/[Lycopene]=75/6.16=12.17.

Binding Experiments of FIG. 5B:

To three 15 mL centrifuge tubes, 200 μL of 4× SDM, 125 μL, or 250 μL or500 μL (for the three tubes) of 9.8 μM Lycopene/HSA solution, and 5575μL or 5450 μL or 5200 μL of PBS (for the three tubes) were added. Eachcentrifuge tube was vortexed for 1 min and the solution in each tube wasanalyzed in a UV-Vis absorbance spectrometer using 1 cm cuvettes. ThisUV-Vis profile was marked as the “HSA/Lycopene only” spectrum. Theentire solution was then transferred back to the original centrifugetube. To these centrifuge tubes, 100 μL of a pathogen suspended in PBSwere added at 10⁸ CFU/mL. The final concentration of the pathogen ineach tube is 1.7×10⁶ CFU/mL. For “control” samples, 100 μL of PBS wereadded without any microorganism. The centrifuge tubes were wrapped in Alfoil and vortexed for 1 min and incubated with a loose cap in a 37° C.shaker for 30 min. The tubes were then vortexed again for 1 min, andanalyzed in the UV-Vis spectrometer. This UV-Vis profile was marked asthe “add bacteria” spectrum. The entire solution was then transferredback to the original tube and centrifuged for 5 min at 4,000 RPM. Thesupernatant from the centrifuge tubes was carefully aliquoted into thecuvette and analyzed again in the UV-Vis spectrometer. This UV-Visprofile was marked as the “after centrifuge” spectrum.

Example 4: Amphotericin B Incorporated into Albumin

Amphotericin B is slightly soluble in methanol and insoluble in acetone.To form a complex of amphotericin B with albumin, a solution ofAmphotericin B in methanol was diluted in acetone with a 1:2 dilution(dilutions of less than 1:2 did not result in the incorporation ofsignificant amounts of Amphotericin B into albumin). Theacetone/methanol solution was mixed with the aqueous albumin solution,and the acetone and methanol removed from the mixture in a rotavapor.The ligand transferred to albumin (FIG. 3).

FIG. 3.

UV-Vis spectrum of an aqueous solution of Amphotericin-B incorporatedinto bovine serum albumin (BSA).

Example 5: Camptothecin Incorporated into Albumin

The anticancer therapeutic agent camptothecin (CPT) dissolves indichloromethane and small amount of methanol. To form a complex of CPTwith albumin, 30 mg of CPT was dissolved in 75 mL of dichloromethane and10 mL of methanol. The resulting dichloromethane/methanol CPT solutionswere diluted by adding 2 times of acetone in volume (once again,dilutions of less than 2 parts acetone did not result in the formationof albumin-CPT complexes). The clear acetone/dichloromethane/methanolCPT solution was added into the aqueous Albumin solution, and the CPTformed a complex with the albumin as the acetone and dichloromethane wasremoved with a rotavapor (FIG. 4).

FIG. 4.

UV-Vis spectrum of an aqueous solution of Camptothecin incorporated intobovine serum albumin (BSA).

Example 6: The Effect of Organic Solvent in the Production ofHydrophobic Ligand-Albumin Complexes

The effect of the organic solvent used in the preparation of ahydrophobic ligand-albumin complex was tested. Several pure volatile andwater soluble organic solvents such as terahydrofuran, methanol,ethanol, and acetone were tested. Of these, only acetone worked; forother solvents, when the solvent was removed, the hydrophobic ligandprecipitated out instead of transferring to albumin.

Several non-volatile solvents, such as DMSO and DMF (boiling point>150°C.), which were removed with a dialysis bag (molecular weight cutoff of1 KDa), were also tested. However, the hydrophobic ligand precipitatedout instead of transferring to albumin. Without intending to be bound byany particular theory, acetone may be effective as a transfer agent dueto a particular combination of properties (solubility parameter,miscibility with water, presence of polar groups, and the ability toalter albumin conformation) that makes it particularly suitable for thispurpose.

Example 7: Reconstituting Freeze Dried Hydrophobic Ligand-AlbuminComplexes

The effect of freeze drying on hydrophobic ligand-albumin complexes wastested. Lycopene in HSA was prepared as described, freeze dried intosheets, and the dried powder resuspended in solution, and the pH of thesolution raised above 8. The resuspension was further sonicated using an800 W sonicator for 10 minutes. The UV-Vis spectrum of the solutionafter sonication was nearly identical to the starting solution (FIG. 2).

FIG. 2.

UV-Vis spectrum of lycopene in HSA in an aqueous solution, as prepared,or freeze dried and resuspended, with and without sonication.

Example 8: Optical Changes in Lycopene Optical Spectrum Due toAggregation

In most chromophores, changes in the chromophore concentration do notchange the chromophores color—the absorption spectrum does not shift tolower, or higher, wavelengths. However, in some chromophores, opticalexchanges come into play when the concentration of the chromophoreincreases beyond a critical level, and two adjacent chromophores canindulge in various optical interactions.

The absorption spectrum of lycopene was compared in dilute andconcentrated forms. In dilute form, absorption spectrum of lycopene hadthree main peaks at 510, 480 and 450 nm, while in concentrated form, anadditional absorption band at 670 nm and 565 nm were observed (FIG. 11).This may be due to formation of hydrophobic pockets that have a verydifferent optical signature as a result of interaction between moleculesin lycopene aggregates formed in a concentrated solution. Further, thecreation of the red shifted absorption bands was accompanied by aredistribution of available vibrational states, i.e., the shape of the450-510 nm triplet changes.

FIG. 11. UV-Vis Absorption Spectrum of Two Lycopene Solutions in Hexane.

When care is taken to ensure that they remain in the dilute form,(presumably, when there are no lycopene aggregates), the spectrum wasdominated by a triplet between 450 and 510 nm. In concentrated solutions(presumably, when aggregates are formed) that are subsequently dilutedto about the same level as the dilute solution, there were additionalabsorption bands at 670, and 565, with potentially a weaker band at 530nm. Also, the distribution of vibrational energies in the 450-510 nmtriplet have changed.

A similar result was obtained for lycopene incorporated into the bindingsites in HSA, as depicted in FIG. 12. HSA was added to a solutioncontaining HSA bound at both of the 2 possible binding sites (Sudlow Iand II) by lycopene. The difference spectrum showed a red shifted peakat 540 nm (FIG. 12). This suggests that the binding sites canpotentially coordinate to enable some lycopene-lycopene interaction.Addition of new HSA may have redistributed the lycopene from Sudlow IIto the newly added HSA (thereby reducing the aggregation). Thisabsorption band is interesting because it affords the possibility of asimple diagnostic tool based on the resonant Raman spectrum collectedwith the 532 nm laser without the possibility of any interference fromother absorption bands.

FIG. 12. Changes in the UV-Vis Absorption Spectrum of Overloaded Lyc/HSAUpon Addition of HSA.

The UV-Vis spectra on top are from 2 samples: (1) Lycopene/HSA 0.55/0.43mM in PBS (ie, some HSA has both Sudlow I and Sudlow II binding sitesoccupied). (2) 0.86 mM HSA was then added, and sonicated for 20 minutes,thereby promoting the exchange of lycopene from some Sudlow II bindingsites to unoccupied Sudlow I sites in the newly added HSA. The twoUV-Vis spectra were nearly identical, and the chart on the bottomdepicts the difference between the two spectrum. The difference spectrumclearly reveals features at 532 nm, these features may be ascribed tothe aggregated form of lycopene (which is likely when both binding sitesare occupied).

Thus, aggregation of albumin should also enable the aggregation oflycopene via a coordination of binding sites. This should change theoptical spectrum of lycopene in a similar manner as was observed uponincreasing the concentration of lycopene.

Potentially, other molecules can also be used. For instance, in thecontext of FIG. 12, β-carotene shows an absorption band at 510 nm, whichis blue-shifted compared to the absorption bands of lycopene by about 20nm.

In addition to the red shifting of the optical spectrum of lycopene, anenhanced absorption was observed as the wavelength decreased below 400nm for concentrated lycopene (FIGS. 11 and 12). This enhanced absorptionmay not be due to any absorption band, but may be due to Rayleighscattering from the clumps of albumin.

Raman scattering from the concentrated lycopene was about 10× lessefficient than that from dilute lycopene, probably because of theenhanced Rayleigh scattering.

Example 9: Aggregation on the Bacterial Cell Wall of Hydrophobic LigandsDelivered Via Hydrophobic Ligand-Albumin Complexes

The binding of HSA/lycopene to bacteria, and the accumulation oflycopene on the bacterial cell wall, were characterized by addingbacteria to a solution of HSA/lycopene in phosphate buffer saline (PBS),removing the bacteria (via a centrifuge step), and comparing theconcentrations of lycopene after the centrifuge step with theconcentration before bacteria addition. If some of the lycopeneaccumulated on the bacteria, then the addition and subsequent removal ofthe bacteria would have also removed some of the lycopene.

The measured UV-Vis absorption profiles of the lycopene/HSA complex inPBS before the addition of bacteria, with the added bacteria, and afterthe centrifuge step are illustrated in FIG. 5A for Staphylococcuswarneri, at 1.7×10⁶ CFU/mL and 0.8 μM lycopene, with 4.5% HSA in PBS(this concentration mimicked human serum). The 4 peaks in the absorptionprofile were due to lycopene, with the peak at 350 nm due to the cisform only and the triplet at 440-520 nm due to both cis and trans forms.

As can be seen in FIG. 5B, Profile A (HSA/lycopene only in the “before”state) is greater than Profile C (after bacteria has been added to it,and then removed via a centrifuge step; the “after centrifuge” state).FIG. 6 summarizes the difference between “initial” state (Profile A inFIG. 5B) and the final state (after centrifuge Profile C in FIG. 5B) for3 different concentrations of lycopene. In all cases, some loss of thelycopene was observed. The loss scales with lycopene concentrationindicating that the same amount of albumin is being lost during thecentrifuge step.

FIG. 5A. UV-Vis Absorbance Spectrum of Bacteria Only (Profile A),Lycopene/HSA (Profile B), and the Two Mixed Together (Profile C).

As can be seen, the two parts, when mixed together, have a much greaterbackground absorbance, compared to the sum of the two parts. Thedifference profile can be fitted with a power function of exponent 2,indicating aggregation of the lycopene/HAS that results in enhancedRayleigh scattering.

FIG. 5B. UV-Vis Spectra of the Lycopene/HSA Solution in the “Before”State (Profile A), after Adding 1.7×10⁶ CFU/mL of S. aureus (Profile B)and after the Final Centrifuge (Profile C).

The difference profile indicates that a substantial amount oflycopene/HSA (nearly 20% of the original) is lost during the centrifugestep.

FIG. 6.

Difference between the UV-Vis absorption profiles of initial and finalstates (Profile A-Profile C in FIG. 5B) for 3 different concentrationsof lycopene in HSA for S. aureus at 1.7×10⁶ CFU/mL.

FIG. 7 summarizes the maximum in this UV-VIS difference plot depicted inFIG. 5B, for 0.8 μM lycopene/HSA for different bacterial and fungalmicroorgansism (all at 1.7×10⁶ CFU/mL), along with the observeddifference in a control sample. Some lycopene loss was observed in thecontrol sample. This is believed to be due to the adsorption of theHSA/lycopene to the centrifuge tube. In all cases, the lycopene loss insamples with the microorganism was significantly greater than that inthe control sample. Thus, it appeared that the transfer and aggregationof lycopene to the microorganism was common to these different types ofmicroorganisms.

FIG. 7. Summary of Binding of HSA to Different Microorganisms.

In the control sample, no bacteria were added, but all other steps(including the centrifuge step) were performed. The decrease in theUV/Vis absorption profile in the control sample was probably due to theadsorption of a small amount of HSA on the centrifuge tube. The decreasein the samples that contains any microorganism was about 4-5× larger.The difference was likely due to the amount of HSA lost because it wasbound to the bacteria which was pelletized by the centrifuge step.

The length of time bacteria was incubated in solution of HSA/lycopene totransfer and aggregate the lycopene to the microorganism cell wallvaried depending on the source of the microorganism. For microorganismssuspended in PBS, 30 minutes was sufficient. For microorganisms in aclinical sample, 10 minutes was sufficient. Thus, the incubation timefor transfer and aggregation of lycopene to the microorganism cell wallmay depend on the whether the microorganism is in a latent state or anactive state.

Example 10: Enhanced Killing Efficacy of Antimicrobials ViaAntimicrobial-Albumin Complexes

In this example, the aggregation of the hydrophobic ligand on thepathogen cell surface was tested using the fungal pathogens Candidaalbicans (ATCC 90028) and C. glabrata (ATCC 2001). The efficacy of thedisclosed formulation of Amphotericin B (Sigma Aldrich catalogue A4888)incorporated into bovine serum albumin (AmpB/BSA) was tested using themethods described herein, with the efficacy of the commerciallyavailable liposomal Amphotericin B (LAMB Sigma Aldrich catalogue A2942).It has been previously reported that the minimum inhibitoryconcentration MIC of Amphotericin B required for inhibiting growth of C.glabrata and C. albicans is about 0.5 μg/mL.

The measurements were set up by calibrating the Amphotericin B contentin the AmpB/BSA formulation described herein, with the commerciallyavailable LAMB formulation. The Amphotericin from both were dissolved inDMSO: H₂O(1:1), and then the UV-Vis absorption curves of AmpB/BSAdissolved in DMSO/H₂O were calibrated with LAMB dissolved in DMSO/H₂O,and then used to calibrate the UV-Vis absorption curve of the aqueoussolution of AmpB/BSA.

The efficacy was demonstrated by comparing the growth of C. albicans andC. glabrata from a starting concentration of 5×10⁵ CFU/mL with varyingamounts of Amphotericin B introduced into the solution as either theAmpB/BSA aqueous solution, or the LAMB formulation. Results are depictedin FIG. 8 for C. albicans and FIG. 9 for C. glabrata. In both cases, thecommercially available LAMB formulation suppressed growth when theconcentration of Amphotericin B exceeded 0.4 μg/mL, which was consistentwith the previously reported MIC values. The AmpB/BSA formulationsuppressed growth when the Amphotericin B concentration exceeded 0.2μg/mL, which corresponded to a very significant reduction of MIC by 2×.This reduction in MIC is consistent with a concentration of thehydrophobic ligand on the cell surface.

FIG. 8. Growth Curves of C. albicans (ATCC 90028) with an InitialConcentration of 5×10⁵ CFU/mL Under Different Concentrations ofAmphotericin B.

LAMB is liposomal Amphotericin B, which is a commercially availablewater soluble form purchased from Sigma (catalogue A2942). AmpB/BSA isthe present water soluble formulation wherein the Amphotericin B issuspended in bovine serum albumin. As can be seen, C. albicans growth issuppressed when the concentration exceeds 0.4 μg/mL for LAMB, and 0.2μg/mL for the disclosed AmpB/BSA formulation.

FIG. 9. Growth Curves of C. glabrata with an Initial Concentration of5×10⁵ CFU/mL Under Different Concentrations of Amphotericin B.

Similar conditions were used as FIG. 8. Once again, the MIC issuppressed from 0.4 μg/mL for LAMB to 0.2 μg/mL for AmpB/BSA.

Example 11: Improved Formulation of Hydrophobic Ligands Via HydrophobicLigand-Albumin Complexes

An albumin based delivery system, as described herein, can be used toexpand the antimicrobial space. There are several existing antimicrobialcompounds with documented in-vitro efficacy when used in an organicsolvent, but which are not used because they are insoluble in water andthe poor solubility poses significant pharmacokinetic challenges. Onesuch example is Clofazimine, which is on the World Health Organization(WHO) list of essential medicines. Clofazimine is an anti-inflammatoryand anti-mycobacterial compound, with an impressive in vitro performanceagainst multidrug-resistant strains of Mycobacterium tuberculosis.However, its use is currently limited to the treatment of leprosybecause it is not water soluble; and thus provides for poorpharmacokinetics against bacteria: Clofazimine is administered as amicrocrystalline suspension in an oil-wax base; and ingestion of a 200mg tablet results in a peak plasma concentration of only 0.41 μg/mL witha time to C_(max) of 8 hours. Since the MIC of this drug against mostgram positive organisms is also about 0.4 μg/mL, the pharmacokinetic(PK) issues prevent its use. Aside from these PK issues, clofazimine isknown to be active against several Gram positive bacteria (via in vitrostudies wherein it is dissolved in DMSO or in acidic ethanol).

To test the feasibility of using albumin as a carrier for Clofazamine,Clofazamine was incorporated in albumin using methods as describeherein, and thus into a water soluble formulation. The in vitro activityof the Clofazamine-albumin complex, water soluble formulation againstStaphylococcus epidermidis is shown in FIG. 10. As shown in the figure,the water soluble formulation had an in vitro activity that was just asgood as the organic formulation. With this formulation, it is possiblethat the PK issues will be addressed by the long retention time ofalbumin in the body.

FIG. 10.

S. Epidermidis in Clofazamine. The two traces represent theconcentration of S. Epidermidis after an 18 hour incubation period, witha starting concentration of 500,000 CFU/mL and a varying concentrationof drug, as indicated on the X-axis. The two traces represent the drugin an organic solvent (2 mg/mL concentration of Clofazamine in 10 mMacetic acid/ethanol; this organic solvent formulation has beenpreviously demonstrated against several gram positive organisms) and thewater soluble formulation wherein Clofazamine was incorporated into BSA.As can be seen, the water formulation affords an MIC of about 0.5 μg/mL,which is identical to that from the organic solvent formulation.

Example 12: Detection of Microorganisms in Clinical Samples ViaHydrophobic Ligand-Albumin Complexes

The following experiments demonstrated that a hydrophobic ligand-albumincomplex of the present disclosure can be used to detect microorganismsin clinical samples.

Detection of Microorganism-Induced Red Shift in the Lycopene OpticalSpectrum.

The hydrophobic ligand-albumin complex-based sensor system includedlycopene (that has been substantially isomerized into the cis form)incorporated into HSA; when a pathogenic microorganism was present inthe vicinity, then the HSA changed conformation. This was observed bycircular dichroism measurements which showed that the distribution ofvibrational energies of the albumin-lycopene complex shifted as a resultof the presence of the microorganism (FIG. 13). While both infected anduninfected samples showed a CD spectra that are dominated by theabsorption triplet of lycopene, in samples that contain a microorganism,the circular dichroism spectrum showed additional absorption bands at565 nm that were not normally seen in the uninfected samples.

FIG. 13.

Circular dichroism spectra of two samples without and with added 1000CFU/mL K. pneumoniae; both prepared with 0.6 ml PHS and 5.4 ml PBS. Inthe infected sample, there is an additional peak at 565 nm, which is dueto aggregation of lycopene.

Interestingly, the UV-Vis absorbance from the lycopene triplet alsochanged with bacteria addition. FIG. 24 summarizes the change in theUV-Vis absorbance when bacteria are added to the HSA/Lycopene. Theprofile included a broad change in the background, consistent with theRayleigh scattering due to the bacteria, but also included features thatresembled the absorbance triplet of lycopene along with a minor peak at565 nm. These features were consistent with transfer and aggregation oflycopene from the HSA complex to the bacteria cell wall—aggregation ofcarotenoids changed the UV-Vis absorbance.

FIG. 24.

Difference between the UV-Vis absorption profiles of initial and finalstates (Profile A-Profile C in FIG. 5B) for 3 different concentrationsof lycopene in HSA for S. aureus at 1.7×10⁶ CFU/mL.

Based on these and other findings, two methods to detect microorganismsin a sample, e.g., clinical sample, were developed: (a) First, a probethat characterizes the optical signature from lycopene was used tocharacterize this signature at a fixed spatial point within the testvial, and as a function of time. As an example, the probe is a Ramanspectrometer using 532 nm illumination, and monitors the lycopene peaksat 1516 and 1156 cm⁻¹. The probe was used to monitor the lycopene Ramanpeaks in the glass vial at a point well above the level at which theaggregated albumin segregates. The energy of the probe is sufficient toalter the conformation of the aggregated albumin—in the examplesprovided herein, 532 nm illumination lasers with powers of 25 mW, 50 mWand 100 mW were used. If pathogenic microorganisms are present, thenthis results in the formation of aggregated albumin, whose conformationis altered by the energy of the incident laser light, which results in asteady decrease of the observed lycopene Raman peaks. Thus, a decreasein the measured lycopene over time is indicative of the presence ofpathogenic microorganisms. (b) The second method involved a probe thatcan move along a linear axis, and which was used to monitor changes inthe spatial profile of the lycopene Raman peak. If any pathogenicmicroorganisms were present in the assay, then this results in thepresence of aggregated albumin (which are not entirely in solution), andthus the spatial profile is not uniform.

Detection of Microorganism-Induced Shift in the Temporal Profile of theLycopene Raman Peak Height

Upon exposure to laser light that is absorbed by the lycopene ligand,with some of this energy being transferred to the host albumin and thusaltering albumin conformation, there was a net decrease in the Ramancross section of lycopene within the aggregates the albumin aggregate(FIG. 15). These changes were reversible, as illustrated in FIG. 15,which plots the lycopene Raman peak height as a function of time at 20mm from the bottom of the test vial. As can be seen in the figure, thelycopene Raman peak height decreased steadily upon laser illumination,and recovers to nearly the original value when the light is turned off.A similar decrease was observed upon subsequent illumination, albeit themagnitude of this decrease was reduced. Because the changes werereversible, they could not be due to any chemical changes, or theformation of any new aggregates of albumin/lycopene. Instead, thesechanges were likely due to photo-induced conformation changes of thehost albumin when it was in the aggregated form. It is believe that thehost albumin in the aggregates rearranged itself so as to enable acoordination of the lycopene binding sites. This coordination results ina red shifting of the optical spectrum of lycopene, and thus a decreasein the Raman peak intensity.

FIG. 15. The Lycopene Peak Height as a Function of Time for a TestSample with 100 CFU/6 mL of S. aureus.

The sample vial was continuously illuminated with laser light (100 mWlaser power, at a fixed position 20 mm from the bottom of the vial) from0-300, 2400-2700, 4800-5100 and 7200-7500 seconds. Upon laserillumination, the lycopene peak height decreases steadily, but recoversto nearly the original value when the illumination is turned off.

The temporal profile of the two dominant Raman peaks was measured whenthe sample was illuminated with 532 nm light. For infected samples, thetime profile of the Raman peak heights showed a decrease over time, withthe changes initiating with laser exposure and saturating out within arelatively short period of time, about 5 to 10 minutes for most samples.As depicted in FIG. 16, the presence of a pathogenic microorganism in asample could be detected by mixing a sample with the lycopene/HSAformulation, and measuring the absolute values of the resonant Ramanpeaks after a 10 min incubation step. These changes weresemi-quantitative, as depicted in FIG. 17. However, the serum from apotentially sick patient will have an unknown (and variable) level oflycopene in it, and so a diagnostic test is developed wherein the rateof change of the Resonant Raman peak can be used as an indicator insteadof the absolute level. One example of this is illustrated in FIG. 18 forS. aureus.

FIG. 16.

The height of the resonant Raman peak at 1156 cm⁻¹ as a function oflycopene content for uninfected samples, and samples that contain 100CFU/mL S. aureus. These measurements were done 20 mm from the bottom ofthe glass vial.

FIG. 17.

Raman peak height as a function of pathogen concentration. All sampleswere 6 mL total, including 500 mL pooled human serum with either 0, or 5or 50 CFUs of added S. aureus (corresponding to pathogen concentrationsof 0, 10 and 100 CFU/mL of PHS).

FIG. 18.

Rate of change of the Raman peaks in 6 samples, each with 6 mL totalsample volume and 500 mL of PHS with varying amounts of S. aureus asindicated.

FIG. 19 illustrates results from a notional diagnostics test that seeksto detect the presence of pathogens in 7 samples that include 3 that areuninfected and 4 infected inoculated with different amounts ofvancomycin resistant enterococci (VRE). As depicted in the figure, thereis a clean separation between uninfected and infected samples using thismethod.

FIG. 19. The negative of the rate of change of the lycopene Raman peakfor 7 samples with varying concentrations of vancomycin resistantEnterococci.

Table 1: Summary of tested microorganisms, the signal average from the 3uninfected control samples, from the 3 infected sampes, and the ratio ofthe pathogen concentrations based on culture and McFarland standards.

TABLE 1 0 10; 100; 1000 Obs/Exp Pathogen CFU/Ml CFU/mL conc. E. coli10418 0.21 −4.36; −4.08; −3.29 0.20 E. coli 12241 0.00 −3.51; −3.07;−1.17 0.18 S. Aureus 0.01 −1.62; −1.97; −1.60 0.44 S. epidermidis −0.18−4.23; −3.85; −3.51 0.71 K. pneumoniae 0.96 −3.41; −3.61; −3.90 0.58 E.cloacae −0.46 −2.03; −1.87; −2.98 0.51 E. faecalis 0.43 −2.56; −2.77;−2.45 0.18 S. pneumoniae −0.35 −5.76; −8.97; −4.37 0.24 P. aeruginosa−0.33 −3.10; −2.16; −3.20 0.70 P. mirabilis 0.86 −9.80; −1.00; −2.401.41 S. pyogenes 0.39 −1.46; −2.59; −2.47 0.95 C. albicans 0.04 −0.86;−1.14; −1.78 2.85 S. typhimurium −0.50 −3.81; −0.60; −2.71 0.45 P.rettgerri 0.94 −1.17; −2.13; −1.06 1.05 B. fragilis −1.51 −1.80; −2.52;−2.86 4.30 A. baumannii 0.78 −2.48; −2.76; −1.63 2.32 E. faecium 0.28−1.87; −4.00; −1.51 1.35 S. maltophilia −0.51 −8.01; −4.40; −1.16 1.23C. glabrata 0.63 −9.17; −2.15; −2.76 0.33 S. aureus (MRSA) 0.13 −2.67;−2.40; −2.08 0.74 C. freundii −0.03 −4.32; −4.94; −2.71 1.96 Average0.09 −3.71; −3.00; −2.46 Stdev 0.60 2.54; 1.78; 0.92

21 different pathogenic microorganisms were tested at clinicallyrelevant concentrations (10 CFU/mL) in a 20 min test, to demonstrate theclinical applicability of the detection method. Results are presented inTable 1, which summarizes the signal output for 21 differentmicroorganisms. In this case, the signal refers to the rate of change ofthe lycopene peak height (all measured 20 mm from the bottom of the testvial), as a function of time. The 0 CFU/mL refers to the average valuefor the 3 uninfected control samples; and Obs/Exp concentration refersto the ratio between the number of viable colonies observed in the testsamples compared to the number expected from the McFarland standard. Insome cases, this ratio was as low as 0.2; the corresponding 10 CFU/mLsample was in fact 2 viable CFU/mL. From the table, it is clear that thesignal from the average uninfected sample was clearly different than thesignal from any of the infected samples.

To gauge the applicability of the disclosed methods to human samples,multiple samples were tested in parallel. In the standard setup that canmeasure 8 samples at a time, 9 experiments spread over 4 days wereperformed, whereby 4 control and 4 infected samples in each experimentwere tested. For all samples, the evolution of the Raman peak wasmonitored for 10 minutes, at a fixed point in the glass vial.

All samples were created with 0.6 mL pooled human serum, and had a totalsample volume of 6 mL containing 0.6 μM lycopene/HSA and 1.5 μMβ-carotene/HSA and had 2 mL of 1× trypticase soy broth (this is added tosupport pathogen viability). In all cases, the pH was controlled to 7.4using a phosphate buffer saline (PBS) with a starting pH of 7.4 and theaddition of a small amount of ascorbic acid. The assay was preparedusing the methods described earlier, and stored in a refrigerator at 5°C. Prior to use, it was incubated in a 37° C. water bath for 30 minutes(this is done so that the albumin conformation reverts to the standardone in the human body), and sonicated using a 100 W ultrasonicator for30 minutes (this is done to break up any aggregates of albumin as it isknown that albumin can form aggregates when it is stored below 37° C.for extended periods).

All the infected samples were created with 0.6 mL pooled human serum andthe addition of 100 CFUs of S. aureus in 0.1 mL of PBS and the controlsamples were created with the addition of 0.6 mL of pooled human serumand 0.1 mL of PBS. The rate of change of the two lycopene peaks at 1516and 1156 cm⁻¹ were measured using 532 nm laser light (at thisillumination wavelength, the Raman spectrum was dominated by thecontribution of lycopene), and FIG. 20 depicts the results of the 9experiments, whereby the data points and error bars represent theaverage and +/−one standard deviation of the 4 control and infectedsamples. It can be seen that the infected samples lie in the red band,and the control sample lie in the blue band.

FIG. 20.

Test results from 9 experiments, each experiment with 4 control and 4infected samples.

Then, detection thresholds established in FIG. 20 were used to test andcharacterize 25 samples created from real human patients and were eitherknown infected or known uninfected depending on the eventual outcome ofthe blood culture testing (in most cases, the blood culture test resultswere not available during the tests). Results are depicted in FIG. 21.In general, there was good concordance between the results and the bloodculture method. The plasma samples were significantly more noisy. Thismay be due to the presence of white blood cells in the plasma samples;in general, febrile patients tend to have a higher white blood cellcount. The two samples that would have been incorrectly diagnosed(sample #s 13 and 16) were excessively noisy due to a very high whiteblood cell count.

It may also be due to the use of potassium phosphate based buffers. Itis known that potassium phosphate reacts with calcium chloride (which isdissolved in the patient plasma) to form potassium chloride and calciumphosphate. Calcium phosphate is insoluble in water, and likely binds tothe albumin. The signal traces are cleaner when all potassium phosphateis removed from the reagents utilized in the disclosed methods. As anexample, buffers were created using HEPES and sodium hydroxide, and theTSB broth was also reformulated using the ingredients and replacingpotassium phosphate with HEPES—these reagents provided for cleanersignal traces.

FIG. 21.

Results from 25 samples using plasma from human patients. In all cases,the “known uninfected” samples are those for which both the bloodculture test (from a different draw on the same patient) and a cultureof the test vial comes in negative, and the “known infected” samples arethose for which either the blood culture, or the culture of the testvial is positive. In some of the “known infected” samples, 100 CFUs ofS. aureus were added to “known uninfected” plasma.

Detection of Microorganism-Induced Shift in the Spatial Profile of theLycopene Raman Peak Height.

It was found that the presence of microorganisms in a sample generates aspatial profile in the Raman peak from lycopene that is distinct fromthat in the absence of the microorganism (FIG. 14). This is consistentwith the aggregates of lycopene-albumin crashing out of solution. Thusaggregation of the lycopene-albumin into a complex that segregates to aseparate layer may be used to detect microorganisms in a sample.

FIG. 14.

The difference between the amplitude of the lycopene Raman peak at 1516cm⁻¹ at time t=0 and t=15 min for a control (uninfected) sample, and onethat contains 100 CFU/6 mL S. aureus. The two plots depict thedifference as a function of distance from the bottom of the glass vial,for a 6 mL test assay that is about 25 mm in height. As shown in thefigure, for the uninfected sample, there is no change in the Raman peakheight, either at the bottom, or at the top of the glass vial. For theinfected sample, the Raman peak height decreases at the top of the glassvial.

The presence of microorganisms in an unknown test sample was diagnosedby characterizing the vertical spatial profile of lycopene (FIGS. 22Aand 22B). The unknown test sample was mixed with lycopene incorporatedinto albumin (lycopene concentration 1.5 μM), as described previously.The mixture was left for 15 minutes for the lycopene-albumin complex tointeract with the bacteria, and tested the samples immediatelyafterwards.

For uninfected samples, because the albumin-lycopene system was insolution, the concentration remained invariant over distance. Forinfected samples, if the albumin had aggregated, then there was a higherconcentration at the bottom of the test vial, as illustrated in FIGS.22A and 22B.

FIGS. 22A and 22B.

(FIG. 22A) The vertical profile of lycopene for two test samples thatboth contain a disclosed assay (with 1 μM lycopene). The profile in bluerepresents an uninfected control sample, and the profile in redrepresents a sample that has 100 CFU of S. aureus in a 6 mL test sample.(FIG. 22B) Slope of the vertical profile, for 4 samples; Samples 1 and 3are uninfected control samples and Samples 2 and 4 have 100 CFU of S.aureus in a 6 mL sample.

Example 13: Diagnosing Antimicrobial Susceptibility Using Lycopene/HSAComplexes

The present lycopene/HSA complexes were used to characterize the minimuminhibitory concentration (MIC) of antimicrobial compounds. Alycopene/HSA complex, prepared as described herein, was mixed with aseries of samples that all had the same initial amount of S. aureus (200CFU/mL of PHS), and which was incubated with a varying amount ofvancomycin for 30 minutes. FIG. 23 shows a plot of the UV-Vis absorbanceat 350 nm against the varying amounts of vancomycin. Upon the additionof the lycopene/HSA assay, aggregation of the lycopene/HSA increased ina manner that scales with S. aureus concentration (and thus on thevancomycin concentration). These changes were characterized via thechanges in the UV-Vis spectrum, depicted at the bottom in FIG. 23. Thechart on the top in FIG. 23 depicts the absorbance at 600 nm for thesesamples after another 18 hours of incubation. This absorbance wasdominated by the pathogen concentration after 18 hours of growth. As canbe seen from the figure, the bottom trace predicted an MIC of about 0.6μg/mL, which is consistent with the chart on top, and also withpreviously reported MIC values for vancomycin/S. aureus, which are inthe range of 0.5 to 1 μg/ml.

FIG. 23. Rapid Prediction of the MIC of S. aureus at 200 CFU/mL.

In this experiment, each 2560 μl sample had 1860 μl of PBS, 200 μl SDM,500 μl of PHS and 600 cfu of added S. aureus, (for an effectiveconcentration of about 234 CFU/mL) along with a variable amount ofvancomycin. Each sample was incubated for 30 min at 37° C. Lycopene(1.125 mM), fucoxanthin (0.375 mM), additional vancomycin (so as tomaintain the same concentration of vancomycin) and additional 3440 μlPBS (to bring the total volume to 6 ml) was added and the UV-Visabsorbance was measured. The chart on the bottom depicts the absorbanceat 450 nm; with the break point representing the predicted MIC of 0.2mg/ml. The chart on the top depicts the absorbance at 600 nm after 18hour incubation at 37° C. The break point of 0.2 μg/ml represents theactual MIC.

This showed that lycopene/HSA complexes can be used to predict theantimicrobial susceptibility of the causative pathogen against ancandidate antimicrobial. Other factors being constant, the signal scaleswith the number of viable pathogenic microorganisms in the sample. For aset of samples that have the same number of microorganisms, a 20 minincubation step with an increasing concentration of a candidateantimicrobial may result in a decreasing number of microorganisms whenthe antimicrobial concentration exceeds the minimum inhibitoryconcentration. Thus, a small incubation step can be combined with thelycopene/HSA complexes to characterize the MIC.

Starting from the initial blood draw, this test for MIC required asample preparation time of about 3 hours (so that the pathogenconcentration can be increased to >1000 CFU/mL; so that the sample couldbe aliquoted into multiple parts with nearly identical pathogenconcentrations), an additional incubation time of 30 minutes and atesting time of less than 5 minutes. Thus, the antimicrobialsusceptibility information could be developed well within 6 hours, andcould be used to influence a 2^(nd) antimicrobial dose.

Example 14: The Molar Ratio of Lycopene to Albumin can Determine Singleor Double Filling of Albumin

Lycopene/albumin complexes were prepared in a similar way as Example 3,except the ratio of lycopene to albumin that were mixed was varied. Whenthe molar ratio of lycopene to albumin was above 0.5, the UV-Visabsorption peaked at 565 nm, an overall red coloration of the solutionwas observed after acetone removal and filtering, and a strongbackground absorption at 600 nm then double filled albumin was observed.This indicated that the albumin was double filled. When the molar ratiowas kept below 0.4, UV-Vis peaks at 565 nm were absent, an overallorange coloration was observed after acetone removal and filtering, andnearly no absorption at 600 nm only single filled albumin was observed.This indicated that the albumin was single filled.

Example 15: Breaking Up Aggregates of Albumin by Sonication Under RaisedpH

In some solutions of the hydrophobic ligand-albumin complexes,aggregation was detected (via an uptick in the absorbance at 600 nm)after a few days of storage at 5° C. This happens at faster rates as thenumber/amount of double filled albumin increased, and if theisomerization of lycopene (from the trans form to the cis form) is notcarried out. In such cases, the nucleation/growth kinetics of aggregatedalbumin is facilitated. But when all the necessary steps to minimizedouble filled albumin are performed, and the cis form is used, storageat room temperature will eventually result in the formation ofaggregated albumin.

For solutions that formed aggregates of hydrophobic ligand-albumincomplexes during storage, the aggregates were broken up by raising thepH of the solutions to above 8.5 and sonicating for a short period (5 to10 minutes), or incubating the complexes at 37° C. for 30 min to 60 min.

While the present disclosure has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of thepresent disclosure. In addition, many modifications may be made to adapta particular situation, material, composition of matter, process,process step or steps, to the objective, spirit and scope of the presentdisclosure. All such modifications are intended to be within the scopeof the claims appended hereto.

What is claimed is:
 1. A method of detecting one or more properties of acarotenoid comprising: dissolving a carotenoid in: i) a first organicsolvent comprising a C₃-C₅ ketone; or ii) a combination of the firstorganic solvent and a second organic solvent in a ratio of from about0.001:1 to about 1000:1 v/v, to provide a first solution; combining thefirst solution with a second solution to provide a third solution,wherein the second solution is an aqueous solution comprising an albuminprotein; and removing the first organic solvent or the combination ofthe first organic solvent and the second organic solvent from the thirdsolution to provide a fourth solution, wherein the fourth solution is anaqueous solution comprising a non-covalent complex of the carotenoid anda single albumin protein, wherein the complex does not include more thanone albumin protein; contacting a microorganism with the fourth solutionto functionally associate the microorganism with the carotenoid; anddetecting one or more properties of the carotenoid.
 2. The method ofclaim 1, wherein the C₃-C₅ ketone is acetone.
 3. The method of claim 1,wherein the carotenoid is dissolved in the combination of a firstorganic solvent and a second organic solvent, and wherein the ratio ofthe first organic solvent and the second organic solvent is in the rangeof about 1:1 to about 5:1.
 4. The method of claim 3, wherein thecarotenoid is dissolved in the combination, and wherein the combinationcomprises the first organic solvent and the second organic solvent in aratio of about 2:1.
 5. The method of claim 1, wherein the carotenoid isdissolved in the combination, and wherein the method comprises:dissolving the carotenoid in the second organic solvent, to provide afifth solution; and combining the fifth solution with the C₃-C₅ ketoneto provide the first solution prior to combining the first solution withthe second solution.
 6. The method of claim 1, wherein the removing isperformed by evaporation.
 7. The method of claim 1, wherein themicroorganism is a pathogenic microorganism.
 8. The method of claim 1,wherein the contacting occurs in vitro.
 9. The method of claim 1,wherein the contacting occurs in vivo.
 10. The method of claim 1,wherein the carotenoid is a carotene.
 11. The method of claim 10,wherein the carotene is lycopene or β-carotene.
 12. The method of claim1, wherein the albumin protein is a human serum albumin protein.
 13. Themethod of claim 1, wherein, the method does not comprise the use of apotassium phosphate containing reagent.